Position dependent recognition of GNN nucleotide triplets by zinc fingers

ABSTRACT

The specificity of binding of a zinc finger to a triplet or quadruplet nucleotide target subsite depends upon the location of the zinc finger in a multifinger protein and, hence, upon the location of its target subsite within a larger target sequence. The present disclosure provides zinc finger amino acid sequences for recognition of triplet target subsites having the nucleotide G in the 5′-most position of the subsite, that have been optimized with respect to the location of the subsite within the target site. Accordingly, the disclosure provides finger position-specific amino acid sequences for the recognition of GNN target subsites. This allows the construction of multi-finger zinc finger proteins with improved affinity and specificity for their target sequences, as well as enhanced biological activity.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent application Ser. No. 09/989,994 filed Nov. 20, 2001 now abandoned which is a continuation-in-part of U.S. patent application Ser. No. 09/535,088 filed Mar. 23, 2000, now abandoned, which application claims the benefit of U.S. provisional applications 60/126,238, filed Mar. 24, 1999, 60/126,239 filed Mar. 24, 1999, 60/146,595 filed Jul. 30, 1999 and 60/146,615 filed Jul. 30, 1999. U.S. patent application Ser. No. 09/989,994 filed Nov. 20, 2001 is also a continuation-in-part of U.S. patent application Ser. No. 09/716,637, filed Nov. 20, 2000, now U.S. Pat. No. 6,794,136. The disclosures of all of the aforementioned applications are hereby incorporated by reference in their entireties for all purposes.

SEQUENCE LISTING

Reference is made to the 947 page sequence listing which accompanies this patent application on compact disc form of 1.68 MB, submitted herewith in duplicate, entitled “Sequence Listing” and created on Sep. 22, 2005, which is herein incorporated by reference.

BACKGROUND

Zinc finger proteins (ZFPs) are proteins that can bind to DNA in a sequence-specific manner. Zinc fingers were first identified in the transcription factor TFIIIA from the oocytes of the African clawed toad, Xenopus laevis. An exemplary motif characterizing one class of these protein (C₂H₂ class) is -Cys-(X)₂₋₄-Cys-(X)₁₂-His-(X)₃₋₅-His (where X is any amino acid) (SEQ. ID. No:1). A single finger domain is about 30 amino acids in length, and several structural studies have demonstrated that it contains an alpha helix containing the two invariant histidine residues and two invariant cysteine residues in a beta turn co-ordinated through zinc. To date, over 10,000 zinc finger sequences have been identified in several thousand known or putative transcription factors. Zinc finger domains are involved not only in DNA-recognition, but also in RNA binding and in protein-protein binding. Current estimates are that this class of molecules will constitute about 2% of all human genes.

The x-ray crystal structure of Zif268, a three-finger domain from a murine transcription factor, has been solved in complex with a cognate DNA sequence and shows that each finger can be superimposed on the next by a periodic rotation. The structure suggests that each finger interacts independently with DNA over 3 base-pair intervals, with side-chains at positions −1, 2, 3 and 6 on each recognition helix making contacts with their respective DNA triplet subsites. The amino terminus of Zif268 is situated at the 3′ end of the DNA strand with which it makes most contacts. Some zinc fingers can bind to a fourth base in a target segment. If the strand with which a zinc finger protein makes most contacts is designated the target strand, some zinc finger proteins bind to a three base triplet in the target strand and a fourth base on the nontarget strand. The fourth base is complementary to the base immediately 3′ of the three base subsite.

The structure of the Zif268-DNA complex also suggested that the DNA sequence specificity of a zinc finger protein might be altered by making amino acid substitutions at the four helix positions (−1, 2, 3 and 6) on each of the zinc finger recognition helices. Phage display experiments using zinc finger combinatorial libraries to test this observation were published in a series of papers in 1994 (Rebar et al., Science 263, 671-673 (1994); Jamieson et al., Biochemistry 33, 5689-5695 (1994); Choo et al, PNAS 91, 11163-11167 (1994)). Combinatorial libraries were constructed with randomized side-chains in either the first or middle finger of Zif268 and then used to select for an altered Zif268 binding site in which the appropriate DNA sub-site was replaced by an altered DNA triplet. Further, correlation between the nature of introduced mutations and the resulting alteration in binding specificity gave rise to a partial set of substitution rules for design of ZFPs with altered binding specificity.

Greisman & Pabo, Science 275, 657-661 (1997) discuss an elaboration of the phage display method in which each finger of a Zif268 was successively randomized and selected for binding to a new triplet sequence. This paper reported selection of ZFPs for a nuclear hormone response element, a p53 target site and a TATA box sequence.

A number of papers have reported attempts to produce ZFPs to modulate particular target sites. For example, Choo et al., Nature 372, 645 (1994), report an attempt to design a ZFP that would repress expression of a bcr-abl oncogene. The target segment to which the ZFPs would bind was a nine base sequence 5′GCA GAA GCC3′ chosen to overlap the junction created by a specific oncogenic translocation fusing the genes encoding bcr and abl. The intention was that a ZFP specific to this target site would bind to the oncogene without binding to abl or bcr component genes. The authors used phage display to screen a mini-library of variant ZFPs for binding to this target segment. A variant ZFP thus isolated was then reported to repress expression of a stably transfected bcr-able construct in a cell line.

Pomerantz et al., Science 267, 93-96 (1995) reported an attempt to design a novel DNA binding protein by fusing two fingers from Zif268 with a homeodomain from Oct-1. The hybrid protein was then fused with a transcriptional activator for expression as a chimeric protein. The chimeric protein was reported to bind a target site representing a hybrid of the subsites of its two components. The authors then constructed a reporter vector containing a luciferase gene operably linked to a promoter and a hybrid site for the chimeric DNA binding protein in proximity to the promoter. The authors reported that their chimeric DNA binding protein could activate expression of the luciferase gene.

Liu et al., PNAS 94, 5525-5530 (1997) report forming a composite zinc finger protein by using a peptide spacer to link two component zinc finger proteins each having three fingers. The composite protein was then further linked to transcriptional activation domain. It was reported that the resulting chimeric protein bound to a target site formed from the target segments bound by the two component zinc finger proteins. It was further reported that the chimeric zinc finger protein could activate transcription of a reporter gene when its target site was inserted into a reporter plasmid in proximity to a promoter operably linked to the reporter.

Choo et al., WO 98/53058, WO98/53059, and WO 98/53060 (1998) discuss selection of zinc finger proteins to bind to a target site within the HIV Tat gene. Choo et al. also discuss selection of a zinc finger protein to bind to a target site encompassing a site of a common mutation in the oncogene ras. The target site within ras was thus constrained by the position of the mutation.

Previously-disclosed methods for the design of sequence-specific zinc finger proteins have often been based on modularity of individual zinc fingers; i.e., the ability of a zinc finger to recognize the same target subsite regardless of the location of the finger in a multi-finger protein. Although, in many instances, a zinc finger retains the same sequence specificity regardless of its location within a multi-finger protein; in certain cases, the sequence specificity of a zinc finger depends on its position. For example, it is possible for a finger to recognize a particular triplet sequence when it is present as finger 1 of a three-finger protein, but to recognize a different triplet sequence when present as finger 2 of a three-finger protein.

Attempts to address situations in which a zinc finger behaves in a non-modular fashion (i.e., its sequence specificity depends upon its location in a multi-finger protein) have, to date, involved strategies employing randomization of key binding residues in multiple adjacent zinc fingers, followed by selection. See, for example, Isalan et al. (2001) Nature Biotechnol. 19:656-660. However, methods for rational design of polypeptides containing non-modular zinc fingers have not heretofore been described.

SUMMARY

The present disclosure provides compositions comprising and methods involving position dependent recognition of GNN nucleotide triplets by zinc fingers.

Thus, provided herein is a zinc finger protein that binds to a target site, said zinc finger protein comprising a first (F1), a second (F2), and a third (F3) zinc finger, ordered F1, F2, F3 from N-terminus to C-terminus, said target site comprising, in 3′ to 5′ direction, a first (S1), a second (S2), and a third (S3) target subsite, each target subsite having the nucleotide sequence GNN, wherein if S1 comprises GAA, F1 comprises the amino acid sequence QRSNLVR (SEQ ID NO:158); if S2 comprises GAA, F2 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801); if S3 comprises GAA, F3 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801); if S1 comprises GAG, F1 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130); if S2 comprises GAG, F2 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130); if S3 comprises GAG, F3 comprises the amino acid sequence RSDNLTR (SEQ ID NO:231); if S1 comprises GAC, F1 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S2 comprises GAC, F2 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S3 comprises GAC, F3 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S1 comprises GAT, F1 comprises the amino acid sequence QSSNLAR (SEQ ID NO:1765); if S2 comprises GAT, F2 comprises the amino acid sequence TSGNLVR (SEQ ID NO:1442); if S3 comprises GAT, F3 comprises the amino acid sequence TSANLSR (SEQ ID NO:377); if S1 comprises GGA, F1 comprises the amino acid sequence QSGHLAR (SEQ ID NO:413); if S2 comprises GGA, F2 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287); if S3 comprises GGA, F3 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287); if S1 comprises GGG, F1 comprises the amino acid sequence RSDHLAR (SEQ ID NO:127); if S2 comprises GGG, F2 comprises the amino acid sequence RSDHLSR (SEQ ID NO:229); if S3 comprises GGG, F3 comprises the amino acid sequence RSDHLSR (SEQ ID NO:229); if S1 comprises GGC, F1 comprises the amino acid sequence DRSHLTR (SEQ ID NO:1506); if S2 comprises GGC, F2 comprises the amino acid sequence DRSHLAR (SEQ ID NO:1092); if S1 comprises GGT, F1 comprises the amino acid sequence QSSHLTR (SEQ ID NO:835); if S2 comprises GGT, F2 comprises the amino acid sequence TSGHLSR (SEQ ID NO:1201); if S3 comprises GGT, F3 comprises the amino acid sequence TSGHLVR (SEQ ID NO:1425); if S1 comprises GCA, F1 comprises the amino acid sequence QSGSLTR (SEQ ID NO:342); if S2 comprises GCA, F2 comprises QSGDLTR (SEQ ID NO:220); if S3 comprises GCA, F3 comprises QSGDLTR (SEQ ID NO:220); if S1 comprises GCG, F1 comprises the amino acid sequence RSDDLTR (SEQ ID NO:188); if S2 comprises GCG, F2 comprises the amino acid sequence RSDDLQR (SEQ ID NO:1844); if S3 comprises GCG, F3 comprises the amino acid sequence RSDDLTR (SEQ ID NO:188); if S1 comprises GCC, F1 comprises the amino acid sequence ERGTLAR (SEQ ID NO:131); if S2 comprises GCC, F2 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417); if S3 comprises GCC, F3 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417); if S1 comprises GCT, F1 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450); if S2 comprises GCT, F2 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450); if S3 comprises GCT, F3 comprises the amino acid sequence QSSDLQR (SEQ ID NO:132); if S1 comprises GTA, F1 comprises the amino acid sequence QSGALTR (SEQ ID NO:1398); if S2 comprises GTA, F2 comprises the amino acid sequence QSGALAR (SEQ ID NO:3339); if S1 comprises GTG, F1 comprises the amino acid sequence RSDALTR (SEQ ID NO:153); if S2 comprises GTG, F2 comprises the amino acid sequence RSDALSR (SEQ ID NO:237); if S3 comprises GTG, F3 comprises the amino acid sequence RSDALTR (SEQ ID NO:153); if S1 comprises GTC, F1 comprises the amino acid sequence DRSALAR (SEQ ID NO:184); if S2 comprises GTC, F2 comprises the amino acid sequence DRSALAR (SEQ ID NO:184); and if S3 comprises GTC, F3 comprises the amino acid sequence DRSALAR (SEQ ID NO:184).

Also provided are methods of designing a zinc finger protein comprising a first (F1), a second (F2), and a third (F3) zinc finger, ordered F1, F2, F3 from N-terminus to C-terminus that binds to a target site comprising, in 3′ to 5′ direction, a first (S1), a second (S2), and a third (S3) target subsite, each target subsite having the nucleotide sequence GNN, the method comprising the steps of (a) selecting the F1 zinc finger such that it binds to the S1 target subsite, wherein if S1 comprises GAA, F1 comprises the amino acid sequence QRSNLVR (SEQ ID NO:158); if S1 comprises GAG, F1 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130); if S1 comprises GAC, F1 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S1 comprises GAT, F1 comprises the amino acid sequence QSSNLAR (SEQ ID NO:1765); if S1 comprises GGA, F1 comprises the amino acid sequence QSGHLAR (SEQ ID NO:413); if S1 comprises GGG, F1 comprises the amino acid sequence RSDHLAR (SEQ ID NO:127); if S1 comprises GGC, F1 comprises the amino acid sequence DRSHLRT (SEQ ID NO:1506); if S1 comprises GGT, F1 comprises the amino acid sequence QSSHLTR (SEQ ID NO:835); if S1 comprises GCA, F1 comprises QSGSLTR (SEQ ID NO:342); if S1 comprises GCG, F1 comprises RSDDLTR (SEQ ID NO:188); if S2 comprises GCG, F2 comprises RSDDLQR (SEQ ID NO:1844); if S1 comprises GCC, F1 comprises ERGTLAR (SEQ ID NO:131); if S1 comprises GCT, F1 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450); if S1 comprises GTA, F1 comprises the amino acid sequence QSGALTR (SEQ ID NO:1398); if S1 comprises GTG, F1 comprises the amino acid sequence RSDALTR (SEQ ID NO:153); if S1 comprises GTC, F1 comprises the amino acid sequence DRSALAR (SEQ ID NO:184); (b) selecting the F2 zinc finger such that it binds to the S2 target subsite, wherein S2 comprises GAA, F2 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801); if S2 comprises GAG, F2 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130); if S2 comprises GAC, F2 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S2 comprises GAT, F2 comprises the amino acid sequence TSGNLVR (SEQ ID NO:1442); if S2 comprises GGA, F2 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287); if S2 comprises GGG, F2 comprises the amino acid sequence RSDHLSR (SEQ ID NO:229); if S2 comprises GGC, F2 comprises the amino acid sequence DRSHLAR (SEQ ID NO:1092); if S2 comprises GGT, F2 comprises the amino acid sequence TSGHLSR (SEQ ID NO:1201); if S2 comprises GCA, F2 comprises the amino acid sequence QSGDLTR (SEQ ID NO:220); if S2 comprises GCC, F2 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417); if S2 comprises GCT, F2 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450); if S2 comprises GTA, F2 comprises the amino acid sequence QSGALAR (SEQ ID NO:3339); if S2 comprises GTG, F2 comprises the amino acid sequence RSDALSR (SEQ ID NO:237); if S2 comprises GTC, F2 comprises the amino acid sequence DRSALAR (SEQ ID NO:184); and (c) selecting the F3 zinc finger such that it binds to the S3 target subsite, wherein if S3 comprises GAA, F3 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801); if S3 comprises GAG, F3 comprises the amino acid sequence RSDNLTR (SEQ ID NO:231); if S3 comprises GAC, F3 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S3 comprises GAT, F3 comprises the amino acid sequence TSANLSR (SEQ ID NO:377); if S3 comprises GGA, F3 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287); if S3 comprises GGG, F3 comprises RSDHLSR (SEQ ID NO:229); if S3 comprises GGT, F3 comprises the amino acid sequence TSGHLVR (SEQ ID NO:1425); if S3 comprises GCA, F3 comprises the amino acid sequence QSGDLTR (SEQ ID NO:220); if S3 comprises GCG, F3 comprises the amino acid sequence RSDDLTR (SEQ ID NO:188); if S3 comprises GCC, F3 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417); if S3 comprises GCT, F3 comprises the amino acid sequence QSSDLQR (SEQ ID NO:132); if S3 comprises GTG, F3 comprises RSDALTR (SEQ ID NO:153); and if S3 comprises GTC, F3 comprises the amino acid sequence DRSALAR (SEQ ID NO:184);

thereby designing a zinc finger protein that binds to a target site.

In certain embodiments of the zinc finger proteins and methods described herein, S1 comprises GAA and F1 comprises the amino acid sequence QRSNLVR (SEQ ID NO:158). In other embodiments, S2 comprises GAA and F2 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801). In other embodiments, S3 comprises GAA and F3 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801). In other embodiments, S1 comprises GAG and F1 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130). In other embodiments, S2 comprises GAG and F2 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130). In other embodiments, S3 comprises GAG and F3 comprises the amino acid sequence RSDNLTR (SEQ ID NO:231). In other embodiments, S1 comprises GAC and F1 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395). In other embodiments, S2 comprises GAC and F2 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395). In other embodiments, S3 comprises GAC and F3 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395). In other embodiments, S1 comprises GAT and F1 comprises the amino acid sequence QSSNLAR (SEQ ID NO:1765). In other embodiments, S2 comprises GAT and F2 comprises the amino acid sequence TSGNLVR (SEQ ID NO:1442). In other embodiments, S3 comprises GAT and F3 comprises the amino acid sequence TSANLSR (SEQ ID NO:377). In other embodiments, S1 comprises GGA and F1 comprises the amino acid sequence QSGHLAR (SEQ ID NO:413). In other embodiments, S2 comprises GGA and F2 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287). In other embodiments, S3 comprises GGA and F3 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287). In other embodiments, S1 comprises GGG and F1 comprises the amino acid sequence RSDHLAR (SEQ ID NO:127). In other embodiments, S2 comprises GGG and F2 comprises the amino acid sequence RSDHLSR (SEQ ID NO:229). In other embodiments, S3 comprises GGG and F3 comprises the amino acid sequence RSDHLSR (SEQ ID NO:229). In other embodiments, S1 comprises GGC and F1 comprises the amino acid sequence DRSHLTR (SEQ ID NO:705). In other embodiments, S2 comprises GGC and F2 comprises the amino acid sequence DRSHLAR (SEQ ID NO:1092). In other embodiments, S1 comprises GGT and F1 comprises the amino acid sequence QSSHLTR (SEQ ID NO:835). In other embodiments, S2 comprises GGT and F2 comprises the amino acid sequence TSGHLSR (SEQ ID NO:1201). In other embodiments, S3 comprises GGT and F3 comprises the amino acid sequence TSGHLVR (SEQ ID NO:1425). In other embodiments, S1 comprises GCA and F1 comprises the amino acid sequence QSGSLTR (SEQ ID NO:342). In other embodiments, S2 comprises GCA and F2 comprises the amino acid sequence QSGDLTR (SEQ ID NO:220). In other embodiments, S3 comprises GCA and F3 comprises the amino acid sequence QSGDLTR (SEQ ID NO:220). In other embodiments, S1 comprises GCG and F1 comprises the amino acid sequence RSDDLTR (SEQ ID NO:188). In other embodiments, S2 comprises GCG and F2 comprises the amino acid sequence RSDDLQR (SEQ ID NO:1844). In other embodiments, S3 comprises GCG and F3 comprises the amino acid sequence RSDDLTR (SEQ ID NO:188). In other embodiments, S1 comprises GCC and F1 comprises the amino acid sequence ERGTLAR (SEQ ID NO:131). In other embodiments, S2 comprises GCC and F2 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417). In other embodiments, S3 comprises GCC and F3 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417). In other embodiments, S1 comprises GCT and F1 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450). In other embodiments, S2 comprises GCT and F2 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450). In other embodiments, S3 comprises GCT and F3 comprises the amino acid sequence QSSDLQR (SEQ ID NO:132). In other embodiments, S1 comprises GTA and F1 comprises the amino acid sequence QSGALTR (SEQ ID NO:1398). In other embodiments, S2 comprises GTA and F2 comprises the amino acid sequence QSGALAR (SEQ ID NO:3339). In other embodiments, S1 comprises GTG and F1 comprises the amino acid sequence RSDALTR (SEQ ID NO:153). In other embodiments, S2 comprises GTG and F2 comprises the amino acid sequence RSDALSR (SEQ ID NO:237). In other embodiments, S3 comprises GTG and F3 comprises the amino acid sequence RSDALTR (SEQ ID NO:153). In other embodiments, S1 comprises GTC and F1 comprises the amino acid sequence DRSALAR (SEQ ID NO:184). In other embodiments, S2 comprises GTC and F2 comprises the amino acid sequence DRSALAR (SEQ ID NO:184). In other embodiments, S3 comprises GTC and F3 comprises the amino acid sequence DRSALAR (SEQ ID NO:184).

Also provided are polypeptides comprising any of zinc finger proteins described herein. In certain embodiments, the polypeptide further comprises at least one functional domain. Also provided are polynucleotides encoding any of the polypeptides described herein. Thus, also provided are nucleic acid encoding zinc fingers, including all of the zinc fingers described above.

Also provided are segments of a zinc finger comprising a sequence of seven contiguous amino acids as shown herein. Also provided are nucleic acids encoding any of these segments and zinc fingers comprising the same.

Also provided are zinc finger proteins comprising first, second and third zinc fingers. The first, second and third zinc fingers comprise respectively first, second and third segments of seven contiguous amino acids as shown herein. Also provided are nucleic acids encoding such zinc finger proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of site selection analysis of two representative zinc finger proteins (leftmost 4 columns) and measurements of binding affinity for each of these proteins to their intended target sequences and to variant target sequences. (rightmost 3 columns). Analysis of ZFP1 is shown in the upper portion of the figure and analysis of ZFP2 is shown in the lower portion of the figure. For the site selection analyses, the amino acid sequences of residues −1 through +6 of the recognition helix of each of the three component zinc fingers (F3 (ZFP1, SEQ ID NO:130; ZFP2, SEQ ID NO:420), F2 (ZFP1, SEQ ID NO:1051; ZFP2, SEQ ID NO:889) and F1 (ZFP1, SEQ ID NO:395; ZFP2, SEQ ID NO:685)) are shown across the top row; the intended target sequence (divided into finger-specific target subsites) is shown across the second row, and a summary of the sequences bound is shown in the third row. Data for F3 is shown in the second column, data for F2 is shown in the third column, and data for F1 is shown in the third column.

For the binding affinity analyses, the designed target sequence for each ZFP (“cognate”) and two related sequences (“Mt”) are shown (column 6), along with the K_(d) for binding of the ZFP to each of these sequences (column 7).

FIG. 2 shows amino acid sequences of zinc finger recognition regions (amino acids −1 through +6 of the recognition helix) that bind to each of the 16 GNN triplet subsites. Three amino acid sequences are shown for each trinucleotide subsite; these correspond to optimal amino acid sequences for recognition of the subsite from each of the three positions (finger 1, F1 (SEQ ID NOS:688, 2534, 676, 1769, 342, 1450, 131, 158, 1765, 395, 1407, 2644, 705, 1398, 1733 & 184); finger 2, F2 (SEQ ID NOS:688, 229, 1446, 3051, 220, 1450, 417, 801, 1442, 395, 1824, 1201, 972, 3339, 1151 & 184); or finger 3, F3 (SEQ ID NOS:943, 229, 676, 1769, 220, 1365, 417, 801, 3525, 395, 1824, 1425, 972, 3592, 952 & 184)) in a three-finger zinc finger protein. Amino acid sequences are from N-terminal to C-terminal; nucleotide sequences are from 5′ to 3′.

Also shown are site selection results for each of the 48 position-dependent GNN-recognizing zinc fingers. These show the number of times a particular nucleotide was present, at a given position, in a collection of oligonucleotide sequences bound by the finger. For example, out of 15 oligonucleotides bound by a zinc finger protein with the amino acid sequence QSGHLAR (SEQ ID NO:413) present at the finger 1 (F1) position, 15 contained a G in the 5′-most position of the subsite, 15 contained a G in the middle position of the subsite, while, at the 3′-most position of the subsite, 10 contained an A, 3 contained a G and 2 contained a T. Accordingly, this particular amino acid sequence is optimal for binding a GGA triplet from the F1 position.

FIGS. 3A, 3B and 3C show site selection data indicating positional dependence of GCA-, GAT- and GGT-binding zinc fingers. The first and fourth (where applicable) rows of each figure show portions of the amino acid sequence of a designed zinc finger protein (F1 column, SEQ ID NOS:220, 1765, 1442, 835 & 1425; F2 column, SEQ ID NOS:220, 1765, 1442, 889, 1425; F3 column, SEQ ID NOS:220, 159, 377, 889 & 1425). Amino acid residues −1 through +6 of each α-helix are listed from left to right. The second and fifth (where applicable) rows show the target sequence, divided into three triplet subsites, one for each finger of the protein shown in the first and fourth (where applicable) rows, respectively. The third and sixth (where applicable) rows show the distribution of nucleotides in the oligonucleotides obtained by site selection with the proteins shown in the first and fourth (where applicable) rows, respectively. FIG. 3A shows data for fingers designed to bind GCA; FIG. 3B shows data for fingers designed to bind GAT; FIG. 3C shows data for fingers designed to bind GGT.

FIGS. 4A and 4B show properties of the engineered ZFP EP2C. FIG. 4A shows site selection data. The first row provides the amino acid sequences (F3, SEQ ID NO:1100; F2, SEQ ID NO:237; F1, SEQ ID NO:1450) of residues −1 through +6 of the recognition helices for each of the three zinc fingers of the EP2C protein. The second row shows the target sequence (5′ to 3′); with the distribution of nucleotides in the oligonucleotides obtained by site selection indicated below the target sequence.

FIG. 4B shows in vitro and in vivo assays for the binding specificity of EP2C. The first three columns show in vitro measurements of binding affinity of EP2C to its intended target sequence and several related sequences. The first column gives the name of each sequence (2C0 is the intended target sequence, compare to FIG. 4A). The second column shows the nucleotide sequence of various target sequences, with differences from the intended target sequence (2C0) highlighted. The third column shows the K_(d) (in nM) for binding of EP2C to each of the target sequences. K_(d)s were determined by gel shift assays, using 2-fold dilution series of EP2C. The right side of the figure (fourth column and bar graph) shows relative luciferase activities (normalized to β-galactosidase levels) in stable cell lines in which expression of EP2C is inducible. Cells were co-transfected with a vector containing a luciferase coding region under the transcriptional control of the target sequence shown in the same row of the figure, and a control vector encoding β-galactosidase. Luciferase and β-galactosidase levels were measured after induction of EP2C expression. Triplicate samples were assayed and the standard deviations are shown in the bar graph. pGL3 is a luciferase-encoding vector lacking EP2C target sequences. 3B is another negative control, in which luciferase expression is under transcriptional control of sequences (3B) unrelated to the EP2C target sequence.

DEFINITIONS

A zinc finger DNA binding protein is a protein or segment within a larger protein that binds DNA in a sequence-specific manner as a result of stabilization of protein structure through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.

Zinc finger proteins can be engineered to recognize a selected target sequence in a nucleic acid. Any method known in the art or disclosed herein can be used to construct an engineered zinc finger protein or a nucleic acid encoding an engineered zinc finger protein. These include, but are not limited to, rational design, selection methods (e.g., phage display) random mutagenesis, combinatorial libraries, computer design, affinity selection, use of databases matching zinc finger amino acid sequences with target subsite nucleotide sequences, cloning from cDNA and/or genomic libraries, and synthetic constructions. An engineered zinc finger protein can comprise a new combination of naturally-occurring zinc finger sequences. Methods for engineering zinc finger proteins are disclosed in co-owned WO 00/41566 and WO 00/42219; as well as in WO 98/53057; WO 98/53058; WO 98/53059 and WO 98/53060; the disclosures of which are hereby incorporated by reference in their entireties. Methods for identifying preferred target sequences, and for engineering zinc finger proteins to bind to such preferred target sequences, are disclosed in co-owned WO 00/42219.

A designed zinc finger protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data.

A selected zinc finger protein is a protein not found in nature whose production results primarily from an empirical process such as phage display.

The term naturally-occurring is used to describe an object that can be found in nature as distinct from being artificially produced by man. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring. Generally, the term naturally-occurring refers to an object as present in a non-pathological (undiseased) individual, such as would be typical for the species.

A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it increases the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by up to several kilobases or more and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.

A specific binding affinity between, for example, a ZFP and a specific target site means a binding affinity of at least 1×10⁶ M⁻¹.

The terms “modulating expression” “inhibiting expression” and “activating expression” of a gene refer to the ability of a zinc finger protein to activate or inhibit transcription of a gene. Activation includes prevention of subsequent transcriptional inhibition (i.e., prevention of repression of gene expression) and inhibition includes prevention of subsequent transcriptional activation (i.e., prevention of gene activation). Modulation can be assayed by determining any parameter that is indirectly or directly affected by the expression of the target gene. Such parameters include, e.g., changes in RNA or protein levels, changes in protein activity, changes in product levels, changes in downstream gene expression, changes in reporter gene transcription (luciferase, CAT, beta-galactosidase, GFP (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)); changes in signal transduction, phosphorylation and dephosphorylation, receptor-ligand interactions, second messenger concentrations (e.g., cGMP, cAMP, IP3, and Ca2+), cell growth, neovascularization, in vitro, in vivo, and ex vivo. Such functional effects can be measured by any means known to those skilled in the art, e.g., measurement of RNA or protein levels, measurement of RNA stability, identification of downstream or reporter gene expression, e.g., via chemiluminescence, fluorescence, calorimetric reactions, antibody binding, inducible markers, ligand binding assays; changes in intracellular second messengers such as cGMP and inositol triphosphate (IP3); changes in intracellular calcium levels; cytokine release, and the like.

A “regulatory domain” refers to a protein or a protein subsequence that has transcriptional modulation activity. Typically, a regulatory domain is covalently or non-covalently linked to a ZFP to modulate transcription. Alternatively, a ZFP can act alone, without a regulatory domain, or with multiple regulatory domains to modulate transcription.

A D-able subsite within a target site has the motif 5′NNGK3′ (SEQ ID NO:4084). A target site containing one or more such motifs is sometimes described as a D-able target site. A zinc finger appropriately designed to bind to a D-able subsite is sometimes referred to as a D-able finger. Likewise a zinc finger protein containing at least one finger designed or selected to bind to a target site including at least one D-able subsite is sometimes referred to as a D-able zinc finger protein.

DETAILED DESCRIPTION I. General

Tables 1-5 list a collection of nonnaturally occurring zinc finger protein sequences and their corresponding target sites. The first column of each table is an internal reference number. The second column lists a 9 or 10 base target site bound by a three-finger zinc finger protein, with the target sites listed in 5′ to 3′ orientation. The third column provides SEQ ID NOs for the target site sequences listed in column 2. The fourth, sixth and eighth columns list amino acid residues from the first, second and third fingers, respectively, of a zinc finger protein which recognizes the target sequence listed in the second column. For each finger, seven amino acids, occupying positions −1 to +6 of the finger, are listed. The numbering convention for zinc fingers is defined below. Columns 5, 7 and 9 provide SEQ ID NOs for the amino acid sequences listed in columns 4, 6 and 8, respectively. The final column of each table lists the binding affinity (i.e., the K_(d) in nM) of the zinc finger protein for its target site. Binding affinities are measured as described below.

Each finger binds to a triplet of bases within a corresponding target sequence. The first finger binds to the first triplet starting from the 3′ end of a target site, the second finger binds to the second triplet, and the third finger binds the third (i.e., the 5′-most) triplet of the target sequence. For example, the RSDSLTS finger (SEQ ID NO: 646) of SBS#201 (Table 2) binds to 5′TTG3′, the ERSTLTR finger (SEQ ID NO: 851) binds to 5′GCC3′ and the QRADLRR finger (SEQ ID NO: 1056) binds to 5′GCA3′.

Table 6 lists a collection of consensus sequences for zinc fingers and the target sites bound by such sequences. Conventional one letter amino acid codes are used to designate amino acids occupying consensus positions. The symbol “X” designates a nonconsensus position that can in principle be occupied by any amino acid. In most zinc fingers of the C₂H₂ type, binding specificity is principally conferred by residues −1, +2, +3 and +6. Accordingly, consensus sequence determining binding specificity typically include at least these residues. Consensus sequences are useful for designing zinc fingers to bind to a given target sequence. Residues occupying other positions can be selected based on sequences in Tables 1-5, or other known zinc finger sequences. Alternatively, these positions can be randomized with a plurality of candidate amino acids and screened against one or more target sequences to refine binding specificity or improve binding specificity. In general, the same consensus sequence can be used for design of a zinc finger regardless of the relative position of that finger in a multi-finger zinc finger protein. For example, the sequence RXDNXXR (SEQ ID NO:4060) can be used to design a N-terminal, central or C-terminal finger of three finger protein. However, some consensus sequences are most suitable for designing a zinc finger to occupy a particular position in a multi-finger protein. For example, the consensus sequence RXDHXXQ (SEQ ID NO:4055) is most suitable for designing a C-terminal finger of a three-finger protein.

II. Characteristics of Zinc Finger Proteins

Zinc finger proteins are formed from zinc finger components. For example, zinc finger proteins can have one to thirty-seven fingers, commonly having 2, 3, 4, 5 or 6 fingers. A zinc finger protein recognizes and binds to a target site (sometimes referred to as a target segment) that represents a relatively small subsequence within a target gene. Each component finger of a zinc finger protein can bind to a subsite within the target site. The subsite includes a triplet of three contiguous bases all on the same strand (sometimes referred to as the target strand). The subsite may or may not also include a fourth base on the opposite strand that is the complement of the base immediately 3′ of the three contiguous bases on the target strand. In many zinc finger proteins, a zinc finger binds to its triplet subsite substantially independently of other fingers in the same zinc finger protein. Accordingly, the binding specificity of zinc finger protein containing multiple fingers is usually approximately the aggregate of the specificities of its component fingers. For example, if a zinc finger protein is formed from first, second and third fingers that individually bind to triplets XXX, YYY, and ZZZ, the binding specificity of the zinc finger protein is 3′XXX YYY ZZZ5′.

The relative order of fingers in a zinc finger protein from N-terminal to C-terminal determines the relative order of triplets in the 3′ to 5′ direction in the target. For example, if a zinc finger protein comprises from N-terminal to C-terminal first, second and third fingers that individualy bind, respectively, to triplets 5′ GAC3′, 5′GTA3′ and 5″GGC3′ then the zinc finger protein binds to the target segment 3′CAGATGCGG5′. If the zinc finger protein comprises the fingers in another order, for example, second finger, first finger, third finger, then the zinc finger protein binds to a target segment comprising a different permutation of triplets, in this example, 3′ATGCAGCGG5′ (see Berg & Shi, Science 271, 1081-1086 (1996)). The assessment of binding properties of a zinc finger protein as the aggregate of its component fingers may, in some cases, be influenced by context-dependent interactions of multiple fingers binding in the same protein.

Two or more zinc finger proteins can be linked to have a target specificity that is the aggregate of that of the component zinc finger proteins (see e.g., Kim & Pabo, PNAS 95, 2812-2817 (1998)). For example, a first zinc finger protein having first, second and third component fingers that respectively bind to XXX, YYY and ZZZ can be linked to a second zinc finger protein having first, second and third component fingers with binding specificities, AAA, BBB and CCC. The binding specificity of the combined first and second proteins is thus 3′XXXYYYZZZ______AAABBBCCC5′, where the underline indicates a short intervening region (typically 0-5 bases of any type). In this situation, the target site can be viewed as comprising two target segments separated by an intervening segment.

Linkage can be accomplished using any of the following peptide linkers. T G E K P: (SEQ. ID. No:2) (Liu et al., 1997, supra.); (G4S)n (SEQ. ID. No:3) (Kim et al., PNAS 93, 1156-1160 (1996); GGRRGGGS; (SEQ. ID. No:4) LRQRDGERP; (SEQ. ID. No:5) LRQKDGGGSERP; (SEQ. ID. No:6) LRQKD(G3S)2 ERP (SEQ. ID. No:7) Alternatively, flexible linkers can be rationally designed using computer programs capable of modeling both DNA-binding sites and the peptides themselves or by phage display methods. In a further variation, noncovalent linkage can be achieved by fusing two zinc finger proteins with domains promoting heterodimer formation of the two zinc finger proteins. For example, one zinc finger protein can be fused with fos and the other with jun (see Barbas et al., WO 95/119431).

Linkage of two zinc finger proteins is advantageous for conferring a unique binding specificity within a mammalian genome. A typical mammalian diploid genome consists of 3×10⁹ bp. Assuming that the four nucleotides A, C, G, and T are randomly distributed, a given 9 bp sequence is present ˜23,000 times. Thus a ZFP recognizing a 9 bp target with absolute specificity would have the potential to bind to ˜23,000 sites within the genome. An 18 bp sequence is present once in 3.4×10¹⁰ bp, or about once in a random DNA sequence whose complexity is ten times that of a mammalian genome.

A component finger of zinc finger protein typically contains about 30 amino acids and has the following motif (N-C):

(SEQ. ID. No: 8) Cys-(X)₂₋₄-Cys-X.X.X.X.X.X.X.X.X.X.X.X-His-(X)₃₋₅- His                         −1 1 2 3 4 5 6 7

The two invariant histidine residues and two invariant cysteine residues in a single beta turn are co-ordinated through zinc (see, e.g., Berg & Shi, Science 271, 1081-1085 (1996)). The above motif shows a numbering convention that is standard in the field for the region of a zinc finger conferring binding specificity. The amino acid on the left (N-terminal side) of the first invariant His residues is assigned the number +6, and other amino acids further to the left are assigned successively decreasing numbers. The alpha helix begins at residue 1 and extends to the residue following the second conserved histidine. The entire helix is therefore of variable length, between 11 and 13 residues.

The process of designing or selecting a nonnaturally occurring or variant ZFP typically starts with a natural ZFP as a source of framework residues. The process of design or selection serves to define nonconserved positions (i.e., positions −1 to +6) so as to confer a desired binding specificity. One suitable ZFP is the DNA binding domain of the mouse transcription factor Zif268. The DNA binding domain of this protein has the amino acid sequence: YACPVESCDRRFSRSDELTRHIRIHTGQKP (F1) (SEQ. ID No:9) FQCRICMRNFSRSDHLTTHIRTHTGEKP (F2) (SEQ. ID. No:10) FACDICGRKFARSDERKRHTKIHLRQK (F3) SEQ. ID. No:11) and binds to a target 5′ GCG TGG GCG 3′ (SEQ ID No:12).

Another suitable natural zinc finger protein as a source of framework residues is Sp-1. The Sp-1 sequence used for construction of zinc finger proteins corresponds to amino acids 531 to 624 in the Sp-1 transcription factor. This sequence is 94 amino acids in length. The amino acid sequence of Sp-1 is as follows: PGKKKQHICHIQGCGKVYGKTSHLRAHLRWHTGERP FMCTWSYCGKRFTRSDELQRHKRTHTGEKK FACPECPKRFMRSDHLSKHIKTHQNKKG (SEQ. ID. No:13) Sp-1 binds to a target site 5′GGG GCG GGG3′ (SEQ ID No: 14).

An alternate form of Sp-1, an Sp-1 consensus sequence, has the following amino acid sequence: meklrngsgd PGKKKQHACPECGKSFSKSSHLRAHQRTHTGERP YKCPECGKSFSRSDELQRHQRTHTGEKP YKCPECGKSFSRSDHLSKHQRTHQNKKG (SEQ. ID. NO:15) (lower case letters are a leader sequence from Shi & Berg, Chemistry and Biology 1, 83-89. (1995). The optimal binding sequence for the Sp-1 consensus sequence is 5′GGGGCGGGG3′ (SEQ ID NO: 16). Other suitable ZFPs are described below.

There are a number of substitution rules that assist rational design of some zinc finger proteins (see Desjarlais & Berg, PNAS 90, 2256-2260 (1993); Choo & Klug, PNAS 91, 11163-11167 (1994); Desjarlais & Berg, PNAS 89, 7345-7349 (1992); Jamieson et al., supra; Choo et al., WO 98/53057, WO 98/53058; WO 98/53059; WO 98/53060). Many of these rules are supported by site-directed mutagenesis of the three-finger domain of the ubiquitous transcription factor, Sp-1 (Desjarlais and Berg, 1992; 1993). One of these rules is that a 5′ G in a DNA triplet can be bound by a zinc finger incorporating arginine at position 6 of the recognition helix. Another substitution rule is that a G in the middle of a subsite can be recognized by including a histidine residue at position 3 of a zinc finger. A further substitution rule is that asparagine can be incorporated to recognize A in the middle of triplet, aspartic acid, glutamic acid, serine or threonine can be incorporated to recognize C in the middle of triplet, and amino acids with small side chains such as alanine can be incorporated to recognize T in the middle of triplet. A further substitution rule is that the 3′ base of triplet subsite can be recognized by incorporating the following amino acids at position −1 of the recognition helix: arginine to recognize G, glutamine to recognize A, glutamic acid (or aspartic acid) to recognize C, and threonine to recognize T. Although these substitution rules are useful in designing zinc finger proteins they do not take into account all possible target sites. Furthermore, the assumption underlying the rules, namely that a particular amino acid in a zinc finger is responsible for binding to a particular base in a subsite is only approximate. Context-dependent interactions between proximate amino acids in a finger or binding of multiple amino acids to a single base or vice versa can cause variation of the binding specificities predicted by the existing substitution rules.

The technique of phage display provides a largely empirical means of generating zinc finger proteins with a desired target specificity (see e.g., Rebar, U.S. Pat. No. 5,789,538; Choo et al., WO 96/06166; Barbas et al., WO 95/19431 and WO 98/543111; Jamieson et al., supra). The method can be used in conjunction with, or as an alternative to rational design. The method involves the generation of diverse libraries of mutagenized zinc finger proteins, followed by the isolation of proteins with desired DNA-binding properties using affinity selection methods. To use this method, the experimenter typically proceeds as follows. First, a gene for a zinc finger protein is mutagenized to introduce diversity into regions important for binding specificity and/or affinity. In a typical application, this is accomplished via randomization of a single finger at positions −1, +2, +3, and +6, and sometimes accessory positions such as +1, +5, +8 and +10. Next, the mutagenized gene is cloned into a phage or phagemid vector as a fusion with gene III of a filamentous phage, which encodes the coat protein pIII. The zinc finger gene is inserted between segments of gene III encoding the membrane export signal peptide and the remainder of pIII, so that the zinc finger protein is expressed as an amino-terminal fusion with pIII or in the mature, processed protein. When using phagemid vectors, the mutagenized zinc finger gene may also be fused to a truncated version of gene III encoding, minimally, the C-terminal region required for assembly of pIII into the phage particle. The resultant vector library is transformed into E. coli and used to produce filamentous phage which express variant zinc finger proteins on their surface as fusions with the coat protein pIII. If a phagemid vector is used, then the this step requires superinfection with helper phage. The phage library is then incubated with target DNA site, and affinity selection methods are used to isolate phage which bind target with high affinity from bulk phage. Typically, the DNA target is immobilized on a solid support, which is then washed under conditions sufficient to remove all but the tightest binding phage. After washing, any phage remaining on the support are recovered via elution under conditions which disrupt zinc finger-DNA binding. Recovered phage are used to infect fresh E. coli, which is then amplified and used to produce a new batch of phage particles. Selection and amplification are then repeated as many times as is necessary to enrich the phage pool for tight binders such that these may be identified using sequencing and/or screening methods. Although the method is illustrated for pIII fusions, analogous principles can be used to screen ZFP variants as pVIII fusions.

In certain embodiments, the sequence bound by a particular zinc finger protein is determined by conducting binding reactions (see, e.g., conditions for determination of K_(d), infra) between the protein and a pool of randomized double-stranded oligonucleotide sequences. The binding reaction is analyzed by an electrophoretic mobility shift assay (EMSA), in which protein-DNA complexes undergo retarded migration in a gel and can be separated from unbound nucleic acid. Oligonucleotides which have bound the finger are purified from the gel and amplified, for example, by a polymerase chain reaction. The selection (i.e. binding reaction and EMSA analysis) is then repeated as many times as desired, with the selected oligonucleotide sequences. In this way, the binding specificity of a zinc finger protein having a particular amino acid sequence is determined.

Zinc finger proteins are often expressed with a heterologous domain as fusion proteins. Common domains for addition to the ZFP include, e.g., transcription factor domains (activators, repressors, co-activators, co-repressors), silencers, oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members etc.); DNA repair enzymes and their associated factors and modifiers; DNA rearrangement enzymes and their associated factors and modifiers; chromatin associated proteins and their modifiers (e.g. kinases, acetylases and deacetylases); and DNA modifying enzymes (e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases) and their associated factors and modifiers. A preferred domain for fusing with a ZFP when the ZFP is to be used for repressing expression of a target gene is a KRAB repression domain from the human KOX-1 protein (Thiesen et al., New Biologist 2, 363-374 (1990); Margolin et al., Proc. Natl. Acad. Sci. USA 91, 4509-4513 (1994); Pengue et al., Nucl. Acids Res. 22:2908-2914 (1994); Witzgall et al., Proc. Natl. Acad. Sci. USA 91, 4514-4518 (1994). Preferred domains for achieving activation include the HSV VP16 activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962 (1997)) nuclear hormone receptors (see, e.g., Torchia et al., Curr. Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factor kappa B (Bitko & Barik, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt, Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28 (1998)), or artificial chimeric functional domains such as VP64 (Seifpal et al., EMBO J. 11, 4961-4968 (1992)).

An important factor in the administration of polypeptide compounds, such as the ZFPs, is ensuring that the polypeptide has the ability to traverse the plasma membrane of a cell, or the membrane of an intra-cellular compartment such as the nucleus. Cellular membranes are composed of lipid-protein bilayers that are freely permeable to small, nonionic lipophilic compounds and are inherently impermeable to polar compounds, macromolecules, and therapeutic or diagnostic agents. However, proteins and other compounds such as liposomes have been described, which have the ability to translocate polypeptides such as ZFPs across a cell membrane.

For example, “membrane translocation polypeptides” have amphiphilic or hydrophobic amino acid subsequences that have the ability to act as membrane-translocating carriers. In one embodiment, homeodomain proteins have the ability to translocate across cell membranes. The shortest internalizable peptide of a homeodomain protein, Antennapedia, was found to be the third helix of the protein, from amino acid position 43 to 58 (see, e.g., Prochiantz, Current Opinion in Neurobiology 6:629-634 (1996)). Another subsequence, the h (hydrophobic) domain of signal peptides, was found to have similar cell membrane translocation characteristics (see, e.g., Lin et al., J. Biol. Chem. 270:1 4255-14258 (1995)).

Examples of peptide sequences which can be linked to a ZFP, for facilitating uptake of ZFP into cells, include, but are not limited to: an 11 amino acid peptide of the tat protein of HIV; a 20 residue peptide sequence which corresponds to amino acids 84-103 of the p16 protein (see Fahraeus et al., Current Biology 6:84 (1996)); the third helix of the 60-amino acid long homeodomain of Antennapedia (Derossi et al., J. Biol. Chem. 269:10444 (1994)); the h region of a signal peptide such as the Kaposi fibroblast growth factor (K-FGF) h region (Lin et al., supra); or the VP22 translocation domain from HSV (Elliot & O'Hare, Cell 88:223-233 (1997)). Other suitable chemical moieties that provide enhanced cellular uptake may also be chemically linked to ZFPs.

Toxin molecules also have the ability to transport polypeptides across cell membranes. Often, such molecules are composed of at least two parts (called “binary toxins”): a translocation or binding domain or polypeptide and a separate toxin domain or polypeptide. Typically, the translocation domain or polypeptide binds to a cellular receptor, and then the toxin is transported into the cell. Several bacterial toxins, including Clostridium perfringens iota toxin, diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus anthracis toxin, and pertussis adenylate cyclase (CYA), have been used in attempts to deliver peptides to the cell cytosol as internal or amino-terminal fusions (Arora et al., J. Biol. Chem., 268:3334-3341 (1993); Perelle et al., Infect. Immun., 61:5147-5156 (1993); Stenmark et al., J. Cell Biol. 113:1025-1032 (1991); Donnelly et al., PNAS 90:3530-3534 (1993); Carbonetti et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 95:295 (1995); Sebo et al., Infect. Immun. 63:3851-3857 (1995); Klimpel et al., PNAS U.S.A. 89:10277-10281 (1992); and Novak et al., J. Biol. Chem. 267:17186-17193 1992)).

Such subsequences can be used to translocate ZFPs across a cell membrane. ZFPs can be conveniently fused to or derivatized with such sequences. Typically, the translocation sequence is provided as part of a fusion protein. Optionally, a linker can be used to link the ZFP and the translocation sequence. Any suitable linker can be used, e.g., a peptide linker.

III. Position Dependence of Subsite Recognition by Zinc Fingers

A number of the polypeptides disclosed herein have been characterized using the methods disclosed in U.S. Pat. No. 6,794,136; which matured from a grandparent of the present application (the disclosure of which is hereby incorporated by reference in its entirety); in particular with respect to the effect of their position, within a multi-finger protein, on their sequence specificity. The results of these investigations provide a set of zinc finger sequences that are optimized for recognition of certain triplet target subsites whose 5′-most nucleotide is a G (i.e., GNN triplet subsites). Thus, particular zinc finger sequences which recognize each of the GNN triplet subsites, from each position of a three-finger zinc finger protein, are provided. See FIG. 2. It will be clear to those of skill in the art that the optimized, position-specific zinc finger sequences disclosed herein for recognition of GNN target subsites are not limited to use in three-finger proteins. For example, they are also useful in six-finger proteins, which can be made by linkage of two three-finger proteins.

A number of zinc finger amino acid sequences which are reported to bind to target subsites in which the 5′-most nucleotide residue is G (i.e., GNN subsites) have recently been disclosed. Segal et al. (1999) Proc. Natl. Acad. Sci. USA 96:2758-2763; Drier et al. (2000) J. Mol. Biol. 303:489-502; U.S. Pat. No. 6,140,081. These GNN-binding zinc fingers were obtained by selection of finger 2 sequences from phage display libraries of three-finger proteins, in which certain amino acid residues of finger 2 had been randomized. Due to the manner in which they were selected, it is not clear whether these sequences would have the same target subsite specificity if they were present in the F1 and/or F3 positions.

Use of the methods and compositions disclosed herein has now allowed identification of specific zinc finger sequences that bind each of the 16 GNN triplet subsites, and for the first time, provides zinc finger sequences that are optimized for recognition of these triplet subsites in a position-dependent fashion. Moreover, in vivo studies of these optimized designs reveal that the functionality of a ZFP is correlated with its binding affinity to its target sequence. See Example 6, infra.

As a result of the discovery, disclosed herein, that sequence recognition by zinc fingers is position-dependent, it is clear that existing design rules will not, in and of themselves, be applicable to every situation in which it is necessary to construct a sequence-specific ZFP. The results disclosed herein show that many zinc fingers that are constructed based on design rules exhibit the sequence specificity predicted by those design rules only at certain finger positions. The position-specific zinc fingers disclosed herein are likely to function more efficiently in vivo and in cultured cells, with fewer nonspecific effects. Highly specific ZFPs, made using position-specific zinc fingers, will be useful tools in studying gene function and will find broad applications in areas as diverse as human therapeutics and plant engineering.

IV. Production of Zinc Finger Proteins

ZFP polypeptides and nucleic acids encoding the same can be made using routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)). In addition, nucleic acids less than about 100 bases can be custom ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company (http://www.genco.com), ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda, Calif.). Similarly, peptides can be custom ordered from any of a variety of sources, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, inc. (http://www.htibio.com), BMA Biomedicals Ltd (U.K.), Bio.Synthesis, Inc.

Oligonucleotides can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is by either denaturing polyacrylamide gel electrophoresis or by reverse phase HPLC. The sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al., Gene 16:21-26 (1981).

Two alternative methods are typically used to create the coding sequences required to express newly designed DNA-binding peptides. One protocol is a PCR-based assembly procedure that utilizes six overlapping oligonucleotides (FIG. 1). Three oligonucleotides (oligos 1, 3, and 5 in FIG. 1) correspond to “universal” sequences that encode portions of the DNA-binding domain between the recognition helices. These oligonucleotides typically remain constant for all zinc finger constructs. The other three “specific” oligonucleotides (oligos 2, 4, and 6 in FIG. 1) are designed to encode the recognition helices. These oligonucleotides contain substitutions primarily at positions −1, 2, 3 and 6 on the recognition helices making them specific for each of the different DNA-binding domains.

The PCR synthesis is carried out in two steps. First, a double stranded DNA template is created by combining the six oligonucleotides (three universal, three specific) in a four cycle PCR reaction with a low temperature annealing step, thereby annealing the oligonucleotides to form a DNA “scaffold.” The gaps in the scaffold are filled in by high-fidelity thermostable polymerase, the combination of Taq and Pfu polymerases also suffices. In the second phase of construction, the zinc finger template is amplified by external primers designed to incorporate restriction sites at either end for cloning into a shuttle vector or directly into an expression vector.

An alternative method of cloning the newly designed DNA-binding proteins relies on annealing complementary oligonucleotides encoding the specific regions of the desired ZFP. This particular application requires that the oligonucleotides be phosphorylated prior to the final ligation step. This is usually performed before setting up the annealing reactions. In brief, the “universal” oligonucleotides encoding the constant regions of the proteins (oligos 1, 2 and 3 of above) are annealed with their complementary oligonucleotides. Additionally, the “specific” oligonucleotides encoding the finger recognition helices are annealed with their respective complementary oligonucleotides. These complementary oligos are designed to fill in the region which was previously filled in by polymerase in the above-mentioned protocol. The complementary oligos to the common oligos 1 and finger 3 are engineered to leave overhanging sequences specific for the restriction sites used in cloning into the vector of choice in the following step. The second assembly protocol differs from the initial protocol in the following aspects: the “scaffold” encoding the newly designed ZFP is composed entirely of synthetic DNA thereby eliminating the polymerase fill-in step, additionally the fragment to be cloned into the vector does not require amplification. Lastly, the design of leaving sequence-specific overhangs eliminates the need for restriction enzyme digests of the inserting fragment. Alternatively, changes to ZFP recognition helices can be created using conventional site-directed mutagenesis methods.

Both assembly methods require that the resulting fragment encoding the newly designed ZFP be ligated into a vector. Ultimately, the ZFP-encoding sequence is cloned into an expression vector. Expression vectors that are commonly utilized include, but are not limited to, a modified pMAL-c2 bacterial expression vector (New England BioLabs or an eukaryotic expression vector, pcDNA (Promega). The final constructs are verified by sequence analysis.

Any suitable method of protein purification known to those of skill in the art can be used to purify ZFPs (see, Ausubel, supra, Sambrook, supra). In addition, any suitable host can be used for expression, e.g., bacterial cells, insect cells, yeast cells, mammalian cells, and the like.

Expression of a zinc finger protein fused to a maltose binding protein (MBP-ZFP) in bacterial strain JM109 allows for straightforward purification through an amylose column (NEB). High expression levels of the zinc finger chimeric protein can be obtained by induction with IPTG since the MBP-ZFP fusion in the pMal-c2 expression plasmid is under the control of the tac promoter (NEB). Bacteria containing the MBP-ZFP fusion plasmids are inoculated into 2×YT medium containing 10 μM ZnCl2, 0.02% glucose, plus 50 μg/ml ampicillin and shaken at 37° C. At mid-exponential growth IPTG is added to 0.3 mM and the cultures are allowed to shake. After 3 hours the bacteria are harvested by centrifugation, disrupted by sonication or by passage through a french pressure cell or through the use of lysozyme, and insoluble material is removed by centrifugation. The MBP-ZFP proteins are captured on an amylose-bound resin, washed extensively with buffer containing 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5 mM DTT and 50 μM ZnCl2, then eluted with maltose in essentially the same buffer (purification is based on a standard protocol from NEB). Purified proteins are quantitated and stored for biochemical analysis.

The dissociation constants of the purified proteins, e.g., Kd, are typically characterized via electrophoretic mobility shift assays (EMSA) (Buratowski & Chodosh, in Current Protocols in Molecular Biology pp. 12.2.1-12.2.7 (Ausubel ed., 1996)). Affinity is measured by titrating purified protein against a fixed amount of labeled double-stranded oligonucleotide target. The target typically comprises the natural binding site sequence flanked by the 3 bp found in the natural sequence and additional, constant flanking sequences. The natural binding site is typically 9 bp for a three-finger protein and 2×9 bp+intervening bases for a six finger ZFP. The annealed oligonucleotide targets possess a 1 base 5′ overhang which allows for efficient labeling of the target with T4 phage polynucleotide kinase. For the assay the target is added at a concentration of 1 nM or lower (the actual concentration is kept at least 10-fold lower than the expected dissociation constant), purified ZFPs are added at various concentrations, and the reaction is allowed to equilibrate for at least 45 min. In addition the reaction mixture also contains 10 mM Tris (pH 7.5), 100 mM KCl, 1 mM MgCl2, 0.1 mM ZnCl2, 5 mM DTT, 10% glycerol, 0.02% BSA. (NB: in earlier assays poly d(IC) was also added at 10-100 μg/μl.)

The equilibrated reactions are loaded onto a 10% polyacrylamide gel, which has been pre-run for 45 min in Tris/glycine buffer, then bound and unbound labeled target is resolved by electrophoresis at 150V. (alternatively, 10-20% gradient Tris-HCl gels, containing a 4% polyacrylamide stacker, can be used) The dried gels are visualized by autoradiography or phosphorimaging and the apparent Kd is determined by calculating the protein concentration that gives half-maximal binding.

The assays can also include determining active fractions in the protein preparations. Active fractions are determined by stoichiometric gel shifts where proteins are titrated against a high concentration of target DNA. Titrations are done at 100, 50, and 25% of target (usually at micromolar levels).

V. Applications of Engineered Zinc Finger Proteins

ZPFs that bind to a particular target gene, and the nucleic acids encoding them, can be used for a variety of applications. These applications include therapeutic methods in which a ZFP or a nucleic acid encoding it is administered to a subject and used to modulate the expression of a target gene within the subject. See, for example, co-owned WO 00/41566. The modulation can be in the form of repression, for example, when the target gene resides in a pathological infecting microrganisms, or in an endogenous gene of the patient, such as an oncogene or viral receptor, that is contributing to a disease state. Alternatively, the modulation can be in the form of activation when activation of expression or increased expression of an endogenous cellular gene can ameliorate a diseased state. For such applications, ZFPs, or more typically, nucleic acids encoding them are formulated with a pharmaceutically acceptable carrier as a pharmaceutical composition.

Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. (see, e.g., Remington's Pharmaceutical Sciences, 17^(th) ed. 1985)). The ZFPs, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be “nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Formulations suitable for parenteral administration, such as, for example, by intravenous, intramuscular, intradermal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

The dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time. The dose is determined by the efficacy and K_(d) of the particular ZFP employed, the target cell, and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose also is determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular patient

In other applications, ZFPs are used in diagnostic methods for sequence specific detection of target nucleic acid in a sample. For example, ZFPs can be used to detect variant alleles associated with a disease or phenotype in patient samples. As an example, ZFPs can be used to detect the presence of particular mRNA species or cDNA in a complex mixtures of mRNAs or cDNAs. As a further example, ZFPs can be used to quantify copy number of a gene in a sample. For example, detection of loss of one copy of a p53 gene in a clinical sample is an indicator of susceptibility to cancer. In a further example, ZFPs are used to detect the presence of pathological microorganisms in clinical samples. This is achieved by using one or more ZFPs specific to genes within the microorganism to be detected. A suitable format for performing diagnostic assays employs ZFPs linked to a domain that allows immobilization of the ZFP on an ELISA plate. The immobilized ZFP is contacted with a sample suspected of containing a target nucleic acid under conditions in which binding can occur. Typically, nucleic acids in the sample are labeled (e.g., in the course of PCR amplification). Alternatively, unlabelled probes can be detected using a second labelled probe. After washing, bound-labelled nucleic acids are detected.

ZFPs also can be used for assays to determine the phenotype and function of gene expression. Current methodologies for determination of gene function rely primarily upon either overexpression or removing (knocking out completely) the gene of interest from its natural biological setting and observing the effects. The phenotypic effects observed indicate the role of the gene in the biological system.

One advantage of ZFP-mediated regulation of a gene relative to conventional knockout analysis is that expression of the ZFP can be placed under small molecule control. By controlling expression levels of the ZFPs, one can in turn control the expression levels of a gene regulated by the ZFP to determine what degree of repression or stimulation of expression is required to achieve a given phenotypic or biochemical effect. This approach has particular value for drug development. By putting the ZFP under small molecule control, problems of embryonic lethality and developmental compensation can be avoided by switching on the ZFP repressor at a later stage in mouse development and observing the effects in the adult animal. Transgenic mice having target genes regulated by a ZFP can be produced by integration of the nucleic acid encoding the ZFP at any site in trans to the target gene. Accordingly, homologous recombination is not required for integration of the nucleic acid. Further, because the ZFP is trans-dominant, only one chromosomal copy is needed and therefore functional knock-out animals can be produced without backcrossing.

All references cited above are hereby incorporated by reference in their entirety for all purposes.

EXAMPLES Example 1 Initial Design of Zinc Finger Proteins and Determination of Binding Affinity

Initial ZFP designs were based on existing design rules, correspondence regimes and ZFP directories, including those disclosed herein (see Tables 1-5) and also in WO 98/53058; WO 98/530059; WO 98/53060 and co-owned U.S. patent application Ser. No. 09/444,241. See also WO 00/42219. Amino acid sequences were conceptually designed using amino acids 532-624 of the human transcription factor Sp1 as a backbone. Polynucleotides encoding designed ZFPs were assembled using a Polymerase Chain Reaction (PCR)-based procedure that utilizes six overlapping oligonucleotides. PCR products were directly cloned cloning into the Tac promoter vector, pMal-c2 (New England Biolabs, Beverly, Mass.) using the KpnI and BamHI restriction sites. The encoded maltose binding protein-ZFP fusion polypeptides were purified according to the manufacturer's procedures (New England Biolabs, Beverly, Mass.). Binding affinity was measured by gel mobility-shift analysis. All of these procedures are described in detail in co-owned WO 00/41566 and WO 00/42219, as well as in Zhang et al. (2000) J. Biol. Chem. 275:33,850-33,860 and Liu et al. (2001) J. Biol. Chem. 276:11,323-11,334; the disclosures of which are hereby incorporated by reference in their entireties.

Example 2 Optimization of Binding Specificity by Site Selection

Designed ZFPs were tested for binding specificity using site selection methods disclosed in parent application U.S. Ser. No. 09/716,637 filed Nov. 20, 2000, now U.S. Pat. No. 6,794,136. Briefly, designed proteins were incubated with a population of labeled, double-stranded oligonucleotides comprising a library of all possible 9- or 10-nucleotide target sequences. Five nanomoles of labeled oligonucleotides were incubated with protein, at a protein concentration 4-fold above its K_(d) for its target sequence. The mixture was subjected to gel electrophoresis, and bound oligonucleotides were identified by mobility shift, and extracted from the gel. The purified bound oligonucleotides were amplified, and the amplification products were used for a subsequent round of selection. At each round of selection, the protein concentration was decreased by 2 fold. After 3-5 rounds of selection, amplification products were cloned into the TOPO TA cloning vector (Invitrogen, Carlsbad, Calif.), and the nucleotide sequences of approximately 20 clones were determined. The identities of the target sites bound by a designed protein were determined from the sequences and expressed as a compilation of subsite binding sequences.

Example 3 Comparison of Site Selection Results with Binding Affinity

To test the correlation between site selection results and the affinity of binding of a ZFP to various related targets, site selection experiments were conducted on 2 three-finger ZFPs, denoted ZFP1 and ZFP2, and the site selection results were compared with K_(d) measurements obtained from quantitative gel-mobility shift assays using the same ZFPs and target sites. Each ZFP was constructed, based on design rules, to bind to a particular nine-nucleotide target sequence (comprising 3 three-nucleotide subsites), as shown in FIG. 1. Site selection results and affinity measurements are also shown in FIG. 1. The site selection results showed that fingers 1 and 3 of both the ZFP1 and ZFP2 proteins preferentially selected their intended target sequences. However, the second finger of each ZFP preferentially selected subsites other than those to which they were designed to bind (e.g., F2 of ZFP1 was designed to bind TCG, but preferentially selected GTG; F2 of ZFP2 was designed to bind GGT, but preferentially selected GGA).

To confirm the site selection results, binding affinities of ZFP1 and ZFP2 were measured (see Example 1, supra), both to their original target sequences and to new target sequences reflecting the site selection results. For example, the Mt-1 sequence contains two base changes (compared to the original target sequence for ZFP1) which result in a change in the sequence of the finger 2 subsite to GTG, reflecting the preferred finger 2 subsite sequence obtained by site selection. In agreement with the site selection results, binding of ZFP1 to the Mt-1 sequence is approximately 4-fold stronger than its binding to the original target sequence (K_(d) of 12.5 nM compared to a K_(d) of 50 nM, see FIG. 1).

For ZFP2, the specificity of finger 2 for the 3′ base of its target subsite was tested, since, although this finger was designed to bind GGT, site selection indicated that it bound preferentially to GGA. Moreover, the site selection results predicted that finger 2 of ZFP2 would bind with approximately equal affinity to GGT and GGC. Accordingly, target sequences containing GGA (Mt-3) and GGC (Mt-4) at the finger 2 subsite were constructed, and binding affinities of ZFP2 to these target sequences, and to its original target sequence (containing GGT at the finger 2 subsite), were compared. In complete agreement with the site selection results, ZFP2 exhibited the strongest binding affinity for the target sequence containing GGA at the finger 2 subsite (K_(d) of 0.5 nM, FIG. 1), and its affinity for target sequences containing either GGT or GGC at the finger 2 subsite was approximately equal (K_(d) of 1 nM for both targets, FIG. 1). Accordingly, the site selection method, in addition to being useful for iterative optimization of binding specificity, can also be used as a useful indicator of binding affinity.

Example 4 Use of Site Selection to Identify Position-Dependent, GNN-Binding Zinc Fingers

A large number of engineered ZFPs have been evaluated, by site selection, to identify zinc fingers that bind to GNN target subsites. In the course of these studies, it became apparent that the binding specificity of a particular zinc finger sequence is, in some instances, dependent upon the position of the zinc finger in the protein, and hence upon the location of the target subsite within the target sequence. For example, if one wishes to design a three-finger zinc finger protein to bind to a target sequence containing the triplet subsite GAT, it is necessary to know whether this subsite is the first, second or third subsite in the target sequence (i.e., whether the GAT subsite will be bound by the first, second or third finger of the protein). Accordingly, over 110 three-finger zinc finger proteins, containing potential GNN-recognizing zinc fingers in various locations, have been evaluated by site selection experiments. Generally, several zinc finger sequences were designed to recognize each GNN triplet, and each design was tested in each of the F1, F2 and F3 positions through 4 to 6 rounds of selection.

The results of these analyses, shown in FIG. 2, provide optimal position-dependent zinc finger sequences (the sequences shown represent amino acid residues −1 through +6 of the recognition helix portion of the finger) for recognition of the 16 GNN target subsites, as well as site selection results for these GNN-specific zinc fingers. Optimal amino acid sequences for recognition of each GNN subsite from each of three positions (finger 1, finger 2 or finger 3) are thereby provided.

GNG-Binding Finger Designs

The amino acid sequence RSDXLXR (SEQ ID NO:4085) (position −1 to +6 of the recognition helix) was found to be optimal for binding to the four GNG triplets, with Asn⁺³ specifying A as the middle nucleotide; His⁺³ specifying G as the middle nucleotide; Ala⁺³ specifying T as the middle nucleotide; and Asp⁺³ specifying cytosine as the middle nucleotide. At the +5 position, Ala, Thr, Ser, and Gln, were tested, and all showed similar specificity profiles by site selection. Interestingly, and in contrast to a previous report (Swirnoff et al. (1995) Mol. Cell. Biol. 15:2275-2287), site selection results indicated that three naturally-occurring GCG-binding fingers from zif268 and Sp1, having the amino acid sequences RSDELTR (SEQ ID NO:123), RSDELQR (SEQ ID NO:302), and RSDERKR (SEQ ID NO:1100), were not GCG-specific. Rather, each of these fingers selected almost equal numbers of GCG and GTG sequences. Analysis of binding affinity by gel-shift experiments confirmed that finger 3 of zif268, having the sequence RSDERKR (SEQ ID NO:1000), binds GCG and GTG with approximately equal affinity.

Position Dependence of GCA-, GAT-, GGT-, GAA- and GCC-Binding Fingers

Based on existing design rules, the amino acid sequence QSGDLTR (SEQ ID NO:220) (−1 through +6) was tested for its ability to bind the GCA triplet from three positions (F1, F2, and F3) within a three-finger ZFP. FIG. 3A shows that the QSGDLTR (SEQ ID NO:220) sequence bound preferentially to the GCA triplet subsite from the F2 and F3 positions, but not from F1. In fact, the presence of QSGDLTR (SEQ ID NO:220) at the F1 position of three different three-finger ZFPs resulted predominantly in selection of GCT. Accordingly, an attempt was made to redesign this sequence to obtain specificity for GCA from the F1 position. Since the sequence Q⁻¹G⁺²S⁺³R⁺⁶ (SEQ ID NO:4065) had previously been selected from a randomized F1 library using GCA as target (Rebar et al. (1994) Science 263:671-673), a D (asp) to S (ser) change was made at the +3 residue of this finger. The resulting sequence, QSGSLTR (SEQ ID NO:342), was tested for its binding specificity by site selection and found to preferentially bind GCA, from the F1 position, in three different ZFPs (see FIG. 2).

The QSGSLTR (SEQ ID NO:342) zinc finger, optimized for recognition of the GCA subsite from the F1 position, was tested for its selectivity when located at the F2 position. Accordingly, two ZFPs, one containing QSGSLTR (SEQ ID NO:342) at finger 2 and one containing QSGDLTR (SEQ ID NO:220) at finger 2 (both having identical F1 sequences and identical F3 sequences) were tested by site selection. The results indicated that, when used at the F2 position, QSGSLTR (SEQ ID NO:342) bound preferentially to GTA, rather than GCA. Thus, for optimal binding of a GCA triplet subsite from the F1 position, the amino acid sequence QSGSLTR (SEQ ID NO:342) is required; while, for optimal binding of the same subsite sequence from F2 or F3, QSGDLTR (SEQ ID NO:220) should be used. Accordingly, different zinc finger amino acid sequences may be needed to specify a particular triplet subsite sequence, depending upon the location of the subsite within the target sequence and, hence, upon the position of the finger in the protein.

Positional effects were also observed for zinc fingers recognizing GAT and GGT subsites. The zinc finger amino acid sequence QSSNLAR (SEQ ID NO:1765) (−1 through +6) is expected to bind to GAT, based on design rules. However, this sequence selected GAT only from the F1 position, and not from the F2 and F3 positions, from which the sequence GAA was preferentially bound (FIG. 3B). Similarly, the amino acid sequence QSSHLTR (SEQ ID NO:835) which, based on design rules, should bind GGT, selected GGT at the F1 position, but not at the F2 and F3 positions, from which it preferentially bound GGA (FIG. 3C). Conversely, the amino acid sequence TSGHLVR (SEQ ID NO:1425) has previously been disclosed to recognize the triplet GGT, based on its selection from a randomized library of zif268 finger 2. U.S. Pat. No. 6,140,081. However, TSGHLVR (SEQ ID NO:1425) was not specific for the GGT subsite when located at the F1 position (FIG. 3C). These results indicate that the binding specificity of many fingers is position dependent, and particularly point out that the sequence specificity of a zinc finger selected from a F2 library may be positionally limited.

The results shown in FIG. 2 indicate that recognition of at least GAA and GCC triplets by zinc fingers is also position dependent.

These positional dependences stand in contrast to earlier published work, which suggested that zinc fingers behaved as independent modules with respect to the sequence specificity of their binding to DNA. Desjarlais et al. (1993) Proc. Natl. Acad. Sci. USA 90:2256-2260.

Example 5 Characterization of EP2C

The engineered zinc finger protein EP2C binds to a target sequence, GCGGTGGCT with a dissociation constant (K_(d)) of 2 nM. Site selection results indicated that fingers 1 and 2 are highly specific for their target subsites, while finger 3 selects GCG (its intended target subsite) and GTG at approximately equal frequencies (FIG. 4A). To confirm these observations, the binding affinities of EP2C to its cognate target sequence, and to variant target sequences, was measured by standard gel-shift analyses (see Example 1, supra). As standards for comparison, the binding affinities of Sp1 and zif268 to their respective targets were also measured under the same conditions, and were determined to be 40 nM for SP1 (target sequence GGGGCGGGG) and 2 nM for zif268 (target sequence GCGTGGGCG). Measurements of binding affinities confirmed that F3 of EP2C bound GTG and GCG equally well (K_(d)s of 2 nM), but bound GAG with a two-fold lower affinity (FIG. 4B). Finger 2 was very specific for the GTG triplet, binding 15-fold less tightly to a GGG triplet (compare 2C0 and 2C3 in FIG. 4B). Finger 1 was also very specific for the GCT triplet, it bound with 4-fold lower affinity to a GAT triplet (2C4) and with 2-fold lower affinity to a GCG triplet (2C5). This example shows, once again, the high degree of correlation between site selection results and binding affinities.

Example 6 Evaluation of Engineered ZFPs by In Vivo Functional Assays

To determine whether a correlation exists between the binding affinity of a engineered ZFP to its target sequence and its functionality in vivo, cell-based reporter gene assays were used to analyze the functional properties of the engineered ZFP EP2C (see Example 5, supra). For these assays, a plasmid encoding the EP2C ZFP, fused to a VP16 transcriptional activation domain, was used to construct a stable cell line (T-Rex-293™, Invitrogen, Carlsbad, Calif.) in which expression of EP2C-VP16 is inducible, as described in Zhang et al., supra. To generate reporter constructs, three tandem copies of the EP2C target site, or its variants (see FIG. 4B, column 2), were inserted between the Mlu I and BglII sites of the pGL3 luciferase-encoding vector (Promega, Madison, Wis.), upstream of the SV40 promoter. Structures of all reporter constructs were confirmed by DNA sequencing.

Luciferase reporter assays were performed by co-transfection of luciferase reporter construct (200 ng) and pCMV-βgal (100 ng, used as an internal control) into the EP2C cells seeded in 6-well plates. Expression of the EP2C-VP16 transcriptional activator was induced with doxycycline (0.05 ug/ml) 24 h after transfection of reporter constructs. Cell lysates were harvested 40 hours post-transfection, luciferase and β-galactosidase activities were measured by the Dual-Light Reporter Assay System (Tropix, Bedford, Mass.), and luciferase activities were normalized to the co-transfected β-galactosidase activities. The results, shown on the right side of FIG. 4B, showed that the normalized luciferase activity for each reporter construct was well correlated with the in vitro binding affinity of EP2C to the target sequence present in the construct. For example, the target sequences to which EP2C bound with greatest affinity (2C0 and 2C2, K_(d) of 2 nM for each) both stimulated the highest levels of luciferase activity, when used to drive luciferase expression in the reporter construct (FIG. 4B). Target sequences to which EP2C bound with 2-fold lower affinity, 2C1 and 2C5 (K_(d) of 4 nM for each), stimulated roughly half the luciferase activity of the 2C0 and 2C2 targets. The 2C3 and 2C4 sequences, for which EP2C showed the lowest in vitro binding affinities, also yielded the lowest levels of in vivo activity when used to drive luciferase expression. Target 3B, a sequence to which EP2C does not bind, yielded background levels of luciferase activity, similar to those obtained with a luciferase-encoding vector lacking EP2C target sequences (pGL3). Thus there exist good correlations between binding affinity (as determined by K_(d) measurement), binding specificity (as determined by site selection) and in vivo functionality for engineered zinc finger proteins.

TABLE 1 SEQ SEQ SEQ SEQ Kd SBS# TARGET ID F1 ID F2 ID F3 ID (nM) 249 GCGGGGGCG 17 RSDELTR 123 RSDHLSR 229 RSDELRR 335 20 250 GCGGGGGCG 18 RSDELTR 124 RSDHLSR 230 RSDTLKK 336 70 251 GCGGAGGCG 19 RSDELTR 125 RSDNLTR 231 RSDELRR 337 27.5 252 GCGGCCGCG 20 RSDELTR 126 DRSSLTR 232 RSDELRR 338 100 253 GGATGGGGG 21 RSDHLAR 127 RSDHLTT 233 QPAHLAR 339 0.75 256 GCGGGGTCC 22 ERGDLTT 128 RSDHLSR 234 RSDELRR 340 800 258 GCGGGCGGG 23 RSDHLTR 129 ERGHLTR 235 RSDELRR 341 15 259 GCAGAGGAG 24 RSDNLAR 130 RSDNLAR 236 QSGSLTR 342 250 261 GAGGTGGCC 25 ERGTLAR 131 RSDALSR 237 RSDNLSR 343 0.5 262 GCGGGGGCT 26 QSSDLQR 132 RSDHLSR 238 RSDELRR 344 20 263 GCGGGGGCT 27 QSSDLQR 133 RSDHLSR 239 RSDTLKK 345 1 264 GTGGCTGCC 28 DRSSLTR 134 QSSDLQR 240 RSDALAR 346 27 265 GTGGCTGCC 29 ERGTLAR 135 QSSDLQR 241 RSDALAR 347 600 269 GGGGCCGGG 30 RSDHLTR 136 DRSSLTR 242 RSDHLTR 348 5 270 GGGGCCGGG 31 RSDHLTR 137 ERGTLAR 243 RSDHLTR 349 52.5 272 GCAGGGGCC 32 DRSSLTR 138 RSDHLSR 244 QSGSLTR 350 20 337 TGCGGGGCAA 33 RSADLTR 139 RSDHLTR 245 ERQHLAT 351 24 338 TGCGGGGCAA 34 RSADLTR 140 RSDHLTR 246 ERDHLRT 352 8 339 TGCGGGGCAA 35 RSADLTR 141 RSDHLTT 247 ERQHLAT 353 64 340 TGCGGGGCAA 36 RSADLTR 142 RSDHLTT 248 ERDHLRT 354 48 341 TGCGGGGCAA 37 RSADLTR 143 RGDHLKD 249 ERQHLAT 355 1000 342 TGCGGGGCAA 38 RSADLTR 144 RGDHLKD 250 ERDHLRT 356 1000 343 TGCGGGGCAA 39 QSGSLTR 145 RSDHLTR 251 ERQHLAT 357 8 344 TGCGGGGCAA 40 QSGSLTR 146 RSDHLTR 252 ERDHLRT 358 6 345 TGCGGGGCAA 41 QSGSLTR 147 RSDHLTT 253 ERQHLAT 359 96 346 TGCGGGGCAA 42 QSGSLTR 148 RSDHLTT 254 ERDHLRT 360 64 347 TGCGGGGCAA 43 QSGSLTR 149 RGDHLKD 255 ERQHLAT 361 1000 348 TGCGGGGCAA 44 QSGSLTR 150 RGDHLKD 256 ERDHLRT 362 1000 367 GGGGGCGGG 45 RSDHLTR 151 DSGHLTR 257 RSDHLQR 363 60 368 GAGGGGGCG 46 RSDELTR 152 RSDHLTR 258 RSDNLTR 364 3.5 369 GTAGTTGTG 47 RSDALTR 153 TGGSLAR 259 QSGSLTR 365 95 370 GTAGTTGTG 48 RSDALTR 154 NRATLAR 260 QSASLTR 366 300 371 GTAGTTGTG 49 RSDALTR 155 NRATLAR 261 QSGSLTR 367 175 372 GTAGTTGTG 50 RSDSLLR 156 TGGSLAR 262 QSASLTR 368 112.5 373 GTAGTTGTG 51 RSDSLLR 157 NPATLAR 263 QSASLTR 369 320 374 GCTGAGGAA 52 QRSNLVR 158 RSDNLTR 264 TSSELQR 370 3.3 375 GAGGAAGAT 53 QQSNLAR 159 QSGNLQR 265 RSDNLTR 371 85 401 GTAGTTGTG 54 RSDALTR 160 TGGSLAR 266 QSASLTR 372 80 403 GTAGTTGTG 55 RSDSLLR 161 NRATLAR 267 QSGSLTR 373 750 421 GTAGTTGTG 56 DSDSLLR 162 TGGSLAR 268 QSGSLTR 374 500 422 GTAGTTGTG 57 RSDSLLR 163 TGGSLTR 269 QSGSLTR 375 200 423 GTAGTTGTG 58 RSDALTR 164 TGGSLAR 270 QRSALAR 376 1000 424 GATGCTGAG 59 RSDNLTR 165 TSSELQR 271 TSANLSR 377 100 425 GATGCTGAG 60 RSDNLTR 166 QSSDLQR 272 QQSNLAR 378 25 426 GATGCTGAG 61 RSDNLTR 167 QSSDLQR 273 TSANLSR 379 5.5 427 GCTGAGGAA 62 QRSNLVR 168 RSDNLTR 274 QSSDLQR 380 1 428 GAAGATGAC 63 DSSNLTR 169 QQSNLAR 275 QRSNLVR 381 120 429 GAAGATGAC 64 DSSNLTR 170 TSANLSR 276 QRSNLVR 382 50 430 GATGACGAC 65 EKANLTR 171 DSSNLTR 277 QQSNLAR 383 250 431 GACGACGGC 66 DSGHLTR 172 DRSNLER 278 DSSNLTR 384 100 432 GACGACGGC 67 DSGHLTR 173 DHANLAR 279 DSSNLTR 385 1000 433 GACGACGGC 68 DSGNLTR 174 DHANLAR 280 DSSNLTR 386 1000 434 GACGGCGTA 69 QSASLTR 175 DSGHLTR 281 EKANLTR 387 152.5 435 GACGGCGTA 70 QSASLTR 176 DSGHLTR 282 ERGNLTR 388 150 436 GACGGCGTA 71 QRSALAR 177 DSGHLTR 283 EKANLTR 389 95 437 GACGGCGTA 72 QRSALAR 178 DSGHLTR 284 ERGNLTR 390 117.5 438 GAGGGGGCG 73 RSDELTR 179 RSDHLTT 285 RSDNLTR 391 62.5 440 GCCGAGGTGC 74 RSDSLLR 180 RSKNLQR 286 ERGTLAR 392 40 441 GGTGGAGTCA 75 DSGSLTR 181 QSGHLQR 287 TSGHLTR 393 250 445 GTCGCAGTGA 76 RSDSLRR 182 QSSDLQK 288 DSGSLTR 394 1000 450 GACTTGGTGC 77 RSDTLAR 183 RGDALTS 289 DRSNLTR 395 130 453 GGTGGAGTCA 78 DRSALAR 184 QSGHLQR 290 DSSKLSR 396 150 461 GAGTACTGTA 79 QRSHLTT 185 DRSNLRT 291 RSDNLAR 397 120 463 GTGGAGGAGA 80 RSDNLTR 186 RSDNLAR 292 RSDALAR 398 0.5 464 GTGGAGGAGA 81 RSDNLTR 187 RSDNLAR 293 RSDSLAR 399 0.4 466 CAGGCTGCGC 82 RSDDLTR 188 QSSDLQR 294 RSDNLRE 400 65 467 CAGGCTGCGC 83 RSDELTR 189 QSSDLQR 295 RGDHLKD 401 800 468 CAGGCTGCGC 84 RSDDLTR 190 QSSDLQR 296 RGDHLKD 402 42 469 GAAGAGGTCT 85 DRSALAR 191 RSDNLAR 297 QSGNLTR 403 13.5 472 GAGGTCTGGA 86 RSSHLTT 192 DRSALAR 298 RSDNLAR 404 80 476 GGAGAGGATG 87 TTSNLRR 193 RSDNLAR 299 QSDHLTR 405 80 477 GGAGAGGATG 88 TTSNLRR 194 RSDNLAR 300 QRAHLAR 406 100 478 GGAGAGGATG 89 TTSNLRR 195 RSDNLAR 301 QSGHLRR 407 60 479 GTGGCGGACC 90 DSSNLTR 196 RSDELQR 302 RSDALAR 408 8.5 480 GTGGCGGACC 91 DSSNLTR 197 RADTLRR 303 RSDALAR 409 5 483 GAGGGCGAAG 92 QSANLAR 198 ESSKLKR 304 RSDNLAR 410 130 484 GAGGGCGAAG 93 QSDNLAR 199 ESSKLKR 305 RSDNLAR 411 1000 485 GGAGAGGTTT 94 QSSALAR 200 RSDNLAR 306 QRAHLAR 412 110 487 GGAGAGGTTT 95 NRATLAR 201 RSDNLAR 307 QSGHLAR 413 76.9 488 TGGTAGGGGG 96 RSDHLAR 202 RSDNLTT 308 RSDHLTT 414 35 490 TAGGGGGTGG 97 RSDSLLR 203 RSDHLTR 309 RSDNLTT 415 1.5 503 GCCGAGGTGC 98 RSDSLLR 204 RSDNLAR 310 ERGTLAR 416 50 504 GCCGAGGTGC 99 RSDSLLR 205 RSDNLAR 311 DRSDLTR 417 25 505 GCCGAGGTGC 100 RSDSLLR 206 RSDNLAR 312 DCRDLAR 418 65 526 GCGGGCGGGC 101 RSDHLTR 207 ERGHLTR 313 RSDTLKK 419 8 543 GAGTGTGTGA 102 RSDLLQR 208 MSHHLKE 314 RSDHLSR 420 50 544 GAGTGTGTGA 103 RSDSLLR 209 MSHHLKE 315 RSDNLAR 421 125 545 GAGTGTGTGA 104 RKDSLVR 210 TSDHLAS 316 RSDNLTR 422 32 546 GAGTGTGTGA 105 RSDLLQR 211 MSHHLKT 317 RLDGLRT 423 500 547 GAGTGTGTGA 106 RKDSLVR 212 TSGHLTS 318 RSDNLTR 424 500 548 GAGTGTGTGA 107 RSSLLQR 213 MSHHLKT 319 RSDHLSR 425 500 549 GAGTGTGTGA 108 RSSLLQR 214 MSHHLKE 320 RSDHLSR 426 500 550 GAGTGTGTGA 109 RKDSLVR 215 TKDHLAS 321 RSDNLTR 427 20 551 GAGTGTGTGA 110 RSDLLQR 216 MSHHLKT 322 RSDHLSR 428 50 552 GAGTGTGTGA 111 RKDSLVR 217 MSHHLKT 323 RSDNLTR 429 31 553 GAGTGTGTGA 112 RSDSLLR 218 MSHHLKE 324 RSDNLTR 430 125 554 GAGTGTGTGA 113 RKDSLVR 219 TSDHLAS 325 RSDNLAR 431 62.5 558 TGCGGGGCA 114 QSGDLTR 220 RSDHLTR 326 DSGHLAS 432 21 559 GAGTGTGTGA 115 RSDSLLR 221 TSDHLAS 327 RSDNLAR 433 1000 560 GAGTGTGTGA 116 RSSLLQR 222 MSHHLKT 328 RSDHLSR 434 500 561 GAGTGTGTGA 117 RKDSLVR 223 MSHHLKE 329 RSDNLAR 435 1000 562 GAGTGTGTGA 118 RSDSLLR 224 TSGHLTS 330 RSDNLAR 436 1000 565 GATGCTGAG 119 RSDNLTR 225 TSSELQR 331 QQSNLAR 437 100 567 GAAGATGAC 120 EKANLTR 226 TSANLSR 332 QRSNLVR 438 47.5 568 GATGACGAC 121 EKAIThTR 227 DSSNLTR 333 TSANLSR 439 300 569 GTAGTTGTG 122 RSDSLLR 228 TGGSLAR 334 QRSALTR 440 52

TABLE 2 SEQ SEQ SEQ SEQ Kd SBS# TARGET ID F1 ID F2 ID F3 ID (nM) 201 GCAGCCTTG 441 RSDSLTS 646 ERSTLTR 851 QRADLRR 1056 1000 202 GCAGCCTTG 442 RSDSLTS 647 ERSTLTR 852 QRADLAR 1057 1000 203 GCAGCCTTG 443 RSDSLTS 648 ERSTLTR 853 QPATLRR 1058 1000 204 GCAGCCTTG 444 RSDSLTS 649 ERSTLTR 854 QRATLAR 1059 1000 205 GAGGTAGAA 445 QSANLAR 650 QSATLAR 855 RSDNLSR 1060 80 206 GAGGTAGAA 446 QSANLAR 651 QSAVLAR 856 RSDNLSR 1061 1000 207 GAGTGGTTA 447 QRASLAS 652 RSDHLTT 857 RSDNLAR 1062 70 208 TAGGTCTTA 448 QRASLAS 653 DRSALAR 858 RSDNLAS 1063 1000 209 GGAGTGGTT 449 QSSALAR 654 RSDALAR 859 QRAHLAR 1064 35 210 GGAGTGGTT 450 NRDTLAR 655 RSDALAR 860 QPAHLAR 1065 65 211 GGAGTGGTT 451 QSSALAR 656 RSDALAS 861 QRAHLAR 1066 140 212 GGAGTGGTT 452 NRDTLAR 657 RSDALAS 862 QRAHLAR 1067 400 213 GTTGCTGGA 453 QRAHLAR 658 QSSTLAR 863 QSSALAR 1068 1000 214 GTTGCTGGA 454 QRAHLAR 659 QSSTLAR 864 NRDTLAR 1069 1000 215 GAAGTCTGT 455 NRDHLMV 660 DRSALAR 865 QSANLSR 1070 1000 216 GAAGTCTGT 456 NRDHLTT 661 DRSALAR 866 QSANLSR 1071 1000 217 GAGGTCGTA 457 QRSALAR 662 DRSALAR 867 RSDNLAR 1072 40 219 GATGTTGAT 458 QQSNLAR 663 NRDTLAR 868 NRDNLSR 1073 1000 220 GATGTTGAT 459 QQSNLAR 664 NRDTLAR 869 QQSNLSR 1074 1000 221 GATGAGTAC 460 DRSNLRT 665 RSDNLAR 870 NRDNLAR 1075 1000 222 GATGAGTAC 461 ERSNLRT 666 RSDNLAR 871 NRDNLAR 1076 1000 223 GATGAGTAC 462 DRSNLRT 667 RSDNLAR 872 QQSNLAR 1077 105 224 GATGAGTAC 463 ERSNLRT 668 RSDNLAR 873 QQSNLAR 1078 1000 225 TGGGAGGTC 464 DRSALAR 669 RSDNLAR 874 RSDHLTT 1079 6 226 GCAGCCTTG 465 RGDALTS 670 ERGTLAR 875 QSGSLTR 1080 1000 227 GCAGCCTTG 466 RGDALTV 671 ERGTLAR 876 QSGSLTR 1081 1000 228 GCAGCCTTG 467 RGDALTM 672 ERGTLAR 877 QSGSLTR 1082 1000 229 GCAGCCTTG 468 RGDALTS 673 ERGTLAR 878 RSDELTR 1083 1000 230 GCAGCCTTG 469 RGDALTV 674 ERGTLAR 879 RSDELTR 1084 1000 231 GCAGCCTTG 470 RGDALTM 675 ERGTLAR 880 RSDELTR 1085 1000 232 GGTGTGGTG 471 RSDALTR 676 RSDALAR 881 NRSHLAR 1086 50 233 GGTGTGGTG 472 RSDALTR 677 RSDALAR 882 QASHLAR 1087 100 235 GTAGAGGTG 473 RSDALTR 678 RSDNLAR 883 QRGALAR 1088 80 236 GGGGAGGGG 474 RSDHLAR 679 RSDNLAR 884 RSDHLSR 1089 0.3 237 GGGGAGGCC 475 ERGTLAR 680 RSDNLAR 885 RSDHLSR 1090 0.3 238 GGGGAGGCC 476 ERGTLAR 681 RSDNLQR 886 RSDHLSR 1091 0.8 239 GGCGGGGAG 477 RSDNLTR 682 RSDHLTR 887 DRSHLAR 1092 0.4 240 GCAGGGGAG 478 RSDNLTR 683 RSDHLSR 888 QSGSLTR 1093 1 242 GGGGGTGCT 479 QSSDLRR 684 QSSHLAR 889 RSDHLSR 1094 1 243 GTGGGCGCT 480 QSSDLRR 685 DRSHLAR 890 RSDALAR 1095 75 244 TAAGAAGGG 481 RSDHLAR 686 QSGNLTR 891 QSGNLRT 1096 100 245 TAAGAAGGG 482 RSDHLAR 687 QSANLTR 892 QSGNLRT 1097 235 246 GAAGGGGAG 483 RSDNLAR 688 RSDHLAR 893 QSGNLTR 1098 2 247 GAAGGGGAG 484 RSDNLAR 689 RSDHLAR 894 QSGNLRR 1099 2 276 GCGGCCGCG 485 RSDELTR 690 ERGTLAR 895 RSDERKR 1100 90 277 GCGGCCGCG 486 RSDELTR 691 DRSSLTR 896 RSDERKR 1101 107 278 GCGGCCGCG 487 QSWELTR 692 ERGTLAR 897 RSDERKR 1102 190 279 GCGGCCGCG 488 QSWELTR 693 DRSSLTR 898 RSDERKR 1103 260 280 GCGGCCGCG 489 QSGSLTR 694 ERGTLAR 899 RSDERKR 1104 160 281 GCGGCCGCG 490 QSGSLTR 695 DRSSLTR 900 RSDERKR 1105 225 282 GCAGAAGTG 491 RGDALTR 696 QSANLTR 901 QSADLAR 1106 1000 283 GCAGAAGTG 492 RSDALTR 697 QSGNLTR 902 QSGSLTR 1107 2 284 GCGGCCGCG 493 QSGSLTR 698 RSDHLTT 903 RSDERKR 1108 1000 285 TGTGCGGCC 494 ERGTLAR 699 RSDELTR 904 SRDHLQS 1109 1000 287 GCAGAAGCG 495 RGPDLAR 700 QSANLTR 905 QSGSLTR 1110 1000 288 GCAGAAGCG 496 RGPDLAR 701 QSANLTR 906 QSGSLTR 1111 1000 289 GCAGAAGCG 497 RGPDLAR 702 QSGNLQR 907 QSGSLTR 1112 800 290 GCAGAAGCG 498 RSDELAR 703 QSANLQR 908 QSADLAR 1113 1000 292 GCAGAAGCG 499 RSDELTR 704 QSANLQR 909 QSGSLTR 1114 1000 293 GTGTGCGGC 500 DRSHLTR 705 ERHSLQT 910 RSDALTR 1115 320 296 TGCGCGGCC 501 ERGTLAR 706 RSDELTR 911 DRDHLQS 1116 1000 297 TGCGCGGCC 502 ERGTLAR 707 RSDELRR 912 DRSHLQT 1117 500 298 GCTTAGGCA 503 QTGELRR 708 RSDNLQK 913 TSGDLSR 1118 4000 299 GCTTAGGCA 504 QTSDLRR 709 RSDNLQK 914 QSSDLQR 1119 4000 300 GCTTAGGCA 505 QTADLRR 710 RSDNLQR 915 QSSDLSR 1120 400 301 GCTTAGGCA 506 QSADLRR 711 RSDNLQT 916 QSSDLSR 1121 350 302 GCTTAGGCA 507 QSGSLTR 712 RSDNLQT 917 QSSDLSR 1122 75 303 GCTTAGGCA 508 QTGSLTR 713 RSDNLQT 918 QSSDLSR 1123 135 304 GCTTAGGCA 509 QTADLTR 714 RSDNLQT 919 QSSDLSR 1124 230 305 GCTTAGGCA 510 QTGDLTR 715 RSDNLQT 920 QSSDLSR 1125 230 306 GCTTAGGCA 511 QTASLTR 716 RSDNLQT 921 QSSDLSR 1126 280 307 GAAGAAGCG 512 RSDELRR 717 QSGNLQR 922 QSGNLSR 1127 50.5 308 GAAGAAGCG 513 RSDELRR 718 QSANLQR 923 QSANLQR 1128 1000 309 GGAGATGCC 514 ERSDLRR 719 QSSNLQR 924 QSGHLSR 1129 4000 310 GGAGATGCC 515 DRSDLTR 720 NRDNLQT 925 QSGHLSR 1130 1000 311 GGAGATGCC 516 DRSTLTR 721 NRDNLQR 926 QSGHLSR 1131 170 312 GGAGATGCC 517 ERGTLAR 722 NRDNLQR 927 QSGHLSR 1132 2000 313 GGAGATGCC 518 DRSDLTR 723 QRSNLQR 928 QSGHLSR 1133 1000 314 GGAGATGCC 519 DRSSLTR 724 QSSNLQR 929 QSGHLSR 1134 117.5 315 GGAGATGCC 520 ERGTLAR 725 QSSNLQR 930 QSGHLSR 1135 265 316 GGAGATGCC 521 ERGTLAR 726 QRDNLQR 931 QSGHLSR 1136 3000 318 TAGGAGATGC 522 RSDALTS 727 RSDNLAR 932 RSDNLAS 1137 100 319 GGGGAAGGG 523 KTSHLRA 728 QSGNLSR 933 RSDHLSR 1138 125 320 GGGGAAGGG 524 RSDHLTR 729 QSGNLSR 934 RSDHLSR 1139 5 321 GGCGGAGAT 525 TTSNLRR 730 QSGHLQR 935 DRSHLTR 1140 200 323 GGCGGAGAT 526 TTSNLRR 731 QSGHLQR 936 DRDHLTR 1141 600 324 GGCGGAGAT 527 TTSNLRR 732 QSGHLQR 937 DRDHLTR 1142 200 325 GTATCTGCT 528 NSSDLTR 733 NSDVLTS 938 QSDVLTR 1143 1000 326 GTATCTGTT 529 NSDALTR 734 NSDVLTS 939 QSDVLTR 1144 1000 327 TCTGCTGGG 530 RSDHLTR 735 NSADLTR 940 NSDDLTR 1145 1000 328 TCTGTTGGG 531 RSDHLTR 736 NSSALTS 941 NSDDLTR 1146 1000 349 GGTGTCGCC 532 DCRDLAR 737 DSGSLTR 942 TSGHLTR 1147 1000 350 TCCGAGGGT 533 TSGHLTR 738 RSDNLTR 943 DCRDLTT 1148 332 351 GCTGGTGTC 534 DSGSLTR 739 TSGHLTR 944 TLHTLTR 1149 1000 352 GGAGGGGTG 535 RSDSLLR 740 RSDHLTR 945 QSDHLTR 1150 26 353 GTTGGAGCC 536 DCRDLAR 741 QSDHLTR 946 TSGALTR 1151 1000 354 GAAGAGGAC 537 DSSNLTR 742 RSDNLTR 947 QRSNLVR 1152 28 355 GAAGAGGAC 538 EKANLTR 743 RSDNLTR 948 QRSNLVR 1153 20 356 GGCTGGGCG 539 RSDELRR 744 RSDHLTK 949 DSDHLSR 1154 1000 357 GGCTGGGCG 540 RSDELRR 745 RSDHLTK 950 DSDHLSR 1155 1000 358 GGCTGGGCG 541 RSDELRR 746 RSDHLTK 951 DSSHLSR 1156 225 361 GGGTTTGGG 542 RSDHLTR 747 QSSALTR 952 RSDHLTR 1157 130 363 GGGTTTGGG 543 RSDHLTR 748 QSSVLTR 953 RSDHLTR 1158 200 364 GTGTCCGAAG 544 RSDNLTR 749 DSAVLTT 954 RSDSLTR 1159 1000 365 GGTGCTGGT 545 QASHLTR 750 QASVLTR 955 QASHLTR 1160 600 366 GAGGGTGCT 546 QASVLTR 751 QASHLTR 956 RSDNLTR 1161 1000 367 GGGGGCGGG 547 RSDHLTR 752 DSGHLTR 957 RSDHLQR 1162 60 368 GAGGGGGCG 548 RSDELTR 753 RSDHLTR 958 RSDNLTR 1163 3.5 369 GTAGTTGTG 549 RSDALTR 754 TGGSLAR 959 QSGSLTR 1164 95 370 GTAGTTGTG 550 RSDALTR 755 NRATLAR 960 QSASLTR 1165 300 371 GTAGTTGTG 551 RSDALTR 756 NRATLAR 961 QSGSLTR 1166 175 372 GTAGTTGTG 552 RSDSLLR 757 TGGSLAR 962 QSASLTR 1167 112.5 373 GTAGTTGTG 553 RSDSLLR 758 NRATLAR 963 QSASLTR 1168 320 374 GCTGAGGAA 554 QRSNLVR 759 RSDNLTR 964 TSSELQR 1169 3.3 375 GAGGAAGAT 555 QQSNLAR 760 QSGNLQR 965 RSDNLTR 1170 85 377 GTGTTGGCAG 556 QSGSLTR 761 RGDALTS 966 RSDALTR 1171 89 378 GCCGAGGAGA 557 RSDNLTR 762 RSDNLTR 967 DRSSLTR 1172 31 379 GCCGAGGAGA 558 RSDNLTR 763 RSDNLTR 968 ERGTLAR 1173 3 380 GAGTCGGAAG 559 QSANLAR 764 RSDELTT 969 RSDNLAR 1174 1000 381 GCAGCTGCGC 560 RSDELTR 765 QSSDLQR 970 QSGDLTR 1175 1.5 383 TGGTTGGTAT 561 QSATLAR 766 RGDALTS 971 RSDHLTT 1176 1000 384 GTGGGCTTCA 562 DRSALTT 767 DRSHLAR 972 RSDALAR 1177 60 385 GGGGCGGAGC 563 RSDNLTR 768 RSDTLKK 973 RSDHLSR 1178 1.2 386 GGGGCGGAGC 564 RSDNLTR 769 RSDELQR 974 RSDHLSR 1179 0.4 387 GGCGAGGCAA 565 QSGSLTR 770 RSDNLAR 975 DRSHLAR 1180 2.5 388 GGCGAGGCAA 566 QSGDLTR 771 RSDNLAR 976 DRSHLAR 1181 28 390 GTGGCAGCGG 567 RSDTLKK 772 QSSDLQK 977 RSDALAR 1182 20 392 GTGGCAGCGG 568 RSDELTR 773 QSSDLQK 978 RSDALAR 1183 1000 396 GCGGGAGCAG 569 QSGSLTR 774 QSGHLQR 979 RSDTLKK 1184 18.8 397 GCGGGAGCAG 570 QSGDLTR 775 QSGHLQR 980 RSDTLKK 1185 25 400 TCAGTGGTGG 571 RSDALAR 776 RSDSLAR 981 QSGDLRT 1186 40 405 GCGGCCGCA 572 RSDELTR 777 ERGTLAR 982 RSDERKR 1187 110 406 GCGGCCGCA 573 RSDELTR 778 DRSSLTR 983 RSDERKR 1188 110 407 GCGGCCGCA 574 QSWELTR 779 ERGTLAR 984 RSDERKR 1189 410 408 GCGGCCGCA 575 QSWELTR 780 DRSSLTR 985 RSDERKR 1190 380 409 GCGGCCGCA 576 QSGSLTR 781 ERGTLAR 986 RSDERKR 1191 50 410 GCAGAAGTC 577 RSDALTR 782 QSGNLTR 987 QSGSLTR 1192 3 411 GCGGCCGCA 578 QSGSLTR 783 RSDHLTT 988 RSDERKR 1193 1000 412 GCGTGGGCG 579 QSGSLTR 784 RSDHLTT 989 RSDERKR 1194 5 413 GCGTGGGCA 580 QSGSLTR 785 RSDHLTT 990 RSDERKR 1195 5 414 GCAGAAGCA 581 RSDELTR 786 QSANLQR 991 QSGSLTR 1196 1000 415 GTGTGCGGA 582 DRSHLTR 787 ERHSLQT 992 RSDALTR 1197 1000 416 TGTGCGGCC 583 ERGTLAR 788 RSDELRR 993 DRSHLQT 1198 1000 493 GGGGTGGCGG 584 RSDTLKK 789 RSDSLAR 994 RSDHLSR 1199 300 494 GCCGAGGAGA 585 RSDNLTR 790 RSDNLTR 995 DRSSLTR 1200 90 496 GGTGGTGGC 586 DTSHLRR 791 TSGHLQR 996 TSGHLSR 1201 1000 497 GTTTGCGTC 587 ETASLRR 792 DSAHLQR 997 TSSALSR 1202 1000 498 GAAGAGGCA 588 QTGELRR 793 RSDNLQR 998 QSGNLSR 1203 30 499 GCTTGTGAG 589 RTSNLRR 794 TSSHLQK 999 DTDHLRR 1204 1000 500 GCTTGTGAG 590 RSDNLTR 795 QSSNLQT 1000 DRSHLAR 1205 1000 501 GTGGGGGTT 591 NRATLAR 796 RSDHLSR 1001 RSDALAR 1206 8 502 GGGGTGGGA 592 QSAHLAR 797 RSDALAR 1002 RSDHLSR 1207 60 507 GAGGTAGAGG 593 RSDNLAR 798 QRSALAR 1003 RSDNLAR 1208 10 508 GAGGTAGAGG 594 RSDNLAR 799 QSATLAR 1004 RSDNLAR 1209 10 509 GTCGTGTGGC 595 RSDHLTT 800 RSDALAR 1005 DRSALAR 1210 100 510 GTTGAGGAAG 596 QSGNLAR 801 RSDNLAR 1006 NPATLAR 1211 100 511 GTTGAGGAAG 597 QSGNLAR 802 RSDNLAR 1007 QSSALAR 1212 100 512 GAGGTGGAAG 598 QSGNLAR 803 RSDALAR 1008 RSDNLAR 1213 10 513 GAGGTGGAAG 599 QSANLAR 804 RSDALAR 1009 RSDNLAR 1214 1.5 514 TAGGTGGTGG 600 RSDALTR 805 RSDALAR 1010 RSDNLTT 1215 10 515 TGGGAGGAGT 601 RSDNLTR 806 RSDNLTR 1011 RSDHLTT 1216 0.5 516 GGAGGAGCT 602 TTSELRR 807 QSGHLQR 1012 QSGHLSR 1217 700 517 GGAGCTGGGG 603 RTDHLRR 808 TSSELQR 1013 QSGHLSR 1218 50 518 GGGGGAGGAG 604 QTGHLRR 809 QSGHLQR 1014 RSDHLSR 1219 30 519 GGGGAGGAGA 605 RSDNLAR 810 RSDNLSR 1015 RSDHLSR 1220 0.3 520 GGAGGAGAT 606 TTANLRR 811 QSGHLQR 1016 QSGHLSR 1221 300 521 GCAGCAGGA 607 QTGHLRR 812 QSGELQR 1017 QSGELSR 1222 1000 522 GATGAGGCA 608 QTGELRR 813 RSDNLQR 1018 TSANLSR 1223 200 527 GGGGAGGATC 609 TTSNLRR 814 RSSNLQR 1019 RSDHLSR 1224 2 528 GGGGAGGATC 610 TTSNLRR 815 RSSNLQR 1020 RSDHLSR 1225 10 529 GAGGCTTGGG 611 RTDHLRK 816 TSAELQR 1021 RSSNLSR 1226 1000 531 GCGGAGGCTT 612 TTGELRR 817 RSSNLQR 1022 RSDELSR 1227 160 532 GCGGAGGCTT 613 QSSDLQR 818 RSSNLQR 1023 RSDELSR 1228 100 533 GCGGAGGCTT 614 QSSDLQR 819 RSDNLAR 1024 RSADLSR 1229 7 534 GCGGAGGCTT 615 QSSDLQR 820 RSDNLAR 1025 RSDDLRR 1230 10 535 GCAGCCGGG 616 RTDHLRR 821 ESSDLQR 1026 QSGELSR 1231 1000 538 GCAGAGGCTT 617 QSSDLQR 822 RSDNLAR 1027 QSGSLTR 1232 70 540 TGGGCAGGCC 618 DRSHLTR 823 QSGSLTR 1028 RSDHLTT 1233 55 541 GGGGAGGAT 619 TTSNLRR 824 RSSNLQR 1029 RSDHLSR 1234 3 570 GGGGAAGGCT 620 DSGHLTR 825 QRSNLVR 1030 RSDHLTR 1235 20 571 GTGTGTGTGT 621 RSDSLTR 826 QRSNLVR 1031 RSDSLLR 1236 1000 572 GCATACGTGG 622 RSDSLLR 827 DKGNLQS 1032 QSDDLTR 1237 1000 573 GCATACGTG 623 RSDSLLR 828 DKGNLQS 1033 QSGDLTR 1238 1000 574 TACGTGGGGT 624 RSDHLTR 829 RSDHLTR 1034 DKGNLQT 1239 25 575 TACGTGGGCT 625 DFSHLTR 830 RSDHLTR 1035 DKGNLQT 1240 472 576 GAGGGTGTTG 626 NSDTLAR 831 TSGHLTR 1036 RSDNLTR 1241 200 577 GGAGCGGGGA 627 RSDHLSR 832 RSDELQR 1037 QSDHLTR 1242 200 579 GGGGTTGAGG 628 RSDNLTR 833 NRDTLAR 1038 TSGHLTR 1243 200 580 GGTGTTGGAG 629 QPAHLAR 834 NRDTLAR 1039 TSGHLTR 1244 1000 581 TACGTGGGTT 630 QSSHLTR 835 RSDSLLR 1040 DKGNLQT 1245 382 583 GTAGGGGTTG 631 NSSALTR 836 RSDHLTR 1041 QSASLTR 1246 46 584 GAAGGCGGAG 632 QAGHLTR 837 DKSHLTR 1042 QSGNLTR 1247 1000 585 GAAGGCGGAG 633 QAGHLTR 838 DSGHLTR 1043 QSGNLTR 1248 1000 587 GGGGGTTACG 634 DKGNLQT 839 TSGHLTR 1044 RSDHLSK 1249 500 588 GGGGGGGGGG 635 RSDHLSR 840 RSDHLTR 1045 RSDHLSK 1250 30 589 GGAGTATGCT 636 DSGHLAS 841 QSATLAR 1046 QSDHLTR 1251 1000 595 TGGTTGGTAT 637 QRGSLAR 842 RGDALTR 1047 RSDHLTT 1252 73.3 597 TGGTTGGTA 638 QNSAMRK 843 RGDALTS 1048 RSDHLTT 1253 1000 598 TGGTTGGTA 639 QRGSLAR 844 RDGSLTS 1049 RSDHLTT 1254 1000 599 TGGTTGGTA 640 QNSAMRK 845 RDGSLTS 1050 RSDHLTT 1255 1000 600 GAGTCGGAA 641 QSANLAR 846 RSDELRT 1051 RSDNLAR 1256 206.7 601 GAGTCGGAA 642 RSANLTR 847 RLDGLRT 1052 RSDNLAR 1257 606.7 602 GAGTCGGAA 643 RSANLTR 848 RQDTLVG 1053 RSDNLAR 1258 616.7 603 GAGTCGGAA 644 QSGNLAR 849 RSDELRT 1054 RSDNLAR 1259 166.7 606 GGGGAGGATC 645 TTSNLRR 850 RSDNLQR 1055 RSDHLSR 1260 0.2

TABLE 3 SEQ SEQ SEQ SEQ Kd SBS# TARGET ID F1 ID F2 ID F3 ID (nM) 897 GAGGAGGTGA 1261 RSDALAR 1347 RSDNLAR 1433 RSDNLVR 1519 0.07 828 GCGGAGGACC 1262 EKANLTR 1348 RSDNLAR 1434 RSDERKR 1520 0.1 884 GAGGAGGTGA 1263 RSDSLTR 1349 RSDNLAR 1435 RSDNLVR 1521 0.15 817 GAGGAGGTGA 1264 RSDSLTR 1350 RSDNLAR 1436 RSDNLAR 1522 0.31 666 GCGGAGGCGC 1265 RSDDLTR 1351 RSDNLTR 1437 RSDTLKK 1523 0.5 829 GCGGAGGACC 1266 EKANLTR 1352 RSDNLAR 1438 RSDTLKK 1524 0.52 670 GACGTGGAGG 1267 RSDNLAR 1353 RSDALAR 1439 DRSNLTR 1525 0.57 801 AAGGAGTCGC 1268 RSADLRT 1354 RSDNLAR 1440 RSDNLTQ 1526 0.85 668 GTGGAGGCCA 1269 ERGTLAR 1355 RSDNLAR 1441 RSDALAR 1527 1.13 895 ATGGATTCAG 1270 QSHDLTK 1356 TSGNLVR 1442 RSDALTQ 1528 1.4 799 GGGGGAGCTG 1271 QSSDLQR 1357 QRAHLER 1443 RSDHLSR 1529 1.85 798 GGGGGAGCTG 1272 QSSDLQR 1358 QSGHLQR 1444 RSDHLSR 1530 3 842 GAGGTGGGCT 1273 DRSHLTR 1359 RSDALAR 1445 RSDNLAR 1531 5.4 894 TCAGTGGTAT 1274 QRSALAR 1360 RSDALSR 1446 QSHDLTK 1532 6.15 892 ATGGATTCAG 1275 QSHDLTK 1361 QQSNLVR 1447 RSDALTQ 1533 6.2 888 TCAGTGGTAT 1276 QSSSLVR 1362 RSDALSR 1448 QSHDLTK 1534 14 739 GCGGGCGGGC 1277 RSDHLTR 1363 ERGHLTR 1449 RSDDLRR 1535 16.5 850 CAGGCTGTGG 1278 RSDALTR 1364 QSSDLTR 1450 RSDNLRE 1536 17 797 GCAGAGGCTG 1279 QSSDLQR 1365 RSDNLAR 1451 QSGDLTR 1537 17.5 891 TCAGTGGTAT 1280 QSSSLVR 1366 RSDALSR 1452 QSGSLRT 1538 18.5 887 TCAGTGGTAT 1281 QRSALAR 1367 RSDALSR 1453 QSGDLRT 1539 23.75 672 TCGGACGTGG 1282 RSDALAR 1368 DRSNLTR 1454 RSDELRT 1540 24 836 GGGGAGGCCC 1283 ERGTLAR 1369 RSDNLAR 1455 RSDHLSR 1541 24.25 674 GCGGCGTCGG 1284 RSDELRT 1370 RADTLRR 1456 RSDTLKK 1542 27.5 849 GGGGCCCTGG 1285 RSDALRE 1371 DRSSLTR 1457 RSDHLTQ 1543 29.05 825 GAATGGGCAG 1286 QSGSLTR 1372 RSDHLTT 1458 QSGNLTR 1544 37.3 673 GCGGGTGTCT 1287 DRSALAR 1373 QSSHLAR 1459 RSDTLKK 1545 48.33 848 GGGGAGGCCC 1288 DRSSLTR 1374 RSDNLAR 1460 RSDHLSR 1546 49.5 662 AGAGCGGCAC 1289 QTGSLTR 1375 RSDELQR 1461 QSGHLNQ 1547 50 667 GAGTCGGACG 1290 DRSNLTR 1376 RSDELRT 1462 RSDNLAR 1548 50 803 GCAGCGGCTC 1291 QSSDLQR 1377 RSDELQR 1463 QSGSLTR 1549 57.5 671 TCGGACGAGT 1292 RSDNLAR 1378 DRSNLTR 1464 RSDELRT 1550 64 851 GAGATGGATC 1293 QSSNLQR 1379 RRDVLMN 1465 RLHNLQR 1551 74 804 GCAGCGGCTC 1294 QSSDLQR 1380 RSDDLNR 1466 QSGSLTR 1552 82.5 669 GACGAGTCGG 1295 RSDELRT 1381 RSDNLAR 1467 DRSNLTR 1553 90 682 GCTGCAGGAG 1296 RSDHLAR 1382 QSGDLTR 1468 QSSDLSR 1554 90 845 GAGATGGATC 1297 QSSNLQR 1383 RSDALRQ 1469 RLHNLQR 1555 112.5 663 AGAGCGGCAC 1298 QTGSLTR 1384 RSDELQR 1470 KNWKLQA 1556 115 738 GCGGGGTCCG 1299 ERGTLTT 1385 RSDHLSR 1471 RSDDLRR 1557 120 664 AGAGCGGCAC 1300 QTGSLTR 1386 RADTLRR 1472 ASSRLAT 1558 125 833 GACTAGGACC 1301 EKANLTR 1387 RSDNLTK 1473 DRSNLTR 1559 136 685 GCTGCAGGAG 1302 RSDHLAR 1388 QSGSLTR 1474 QSSDLSR 1560 150 835 TAGGGAGCGT 1303 RADTLRR 1389 QSGHLTR 1475 RSDNLTT 1561 150 847 TAGGGAGCGT 1304 RSDDLTR 1390 QSGHLTR 1476 RSDNLTT 1562 150 818 GAATGGGCAG 1305 QSGSLTR 1391 RSDHLTT 1477 QSSNLVR 1563 167 834 GACTAGGACC 1306 EKANLTR 1392 RSDHLTT 1478 DRSNLTR 1564 186 837 GGGGCCCTGG 1307 RSDALRE 1393 DRSSLTR 1479 RSDHLSR 1565 222 764 GCAGAGGCTG 1308 TSGELVR 1394 RSDNLAR 1480 QSGDLTR 1566 255 774 GCAGCGGTAG 1309 QRSALAR 1395 RSDELQR 1481 QSGDLTR 1567 258 765 GCCGAGGCCG 1310 ERGTLAR 1396 RSDNLAR 1482 ERGTLAR 1568 262.5 766 GCCGAGGCCG 1311 ERGTLAR 1397 RSDNLAR 1483 DRSDLTR 1569 262.5 775 GCAGCGGTAG 1312 QSGALTR 1398 RSDELQR 1484 QSGDLTR 1570 265 763 GCAGAGGCTG 1313 TSGELVR 1399 RSDNLAR 1485 QSGSLTR 1571 275 838 GGGGCCCTGG 1314 RSDALRE 1400 DRSSLTR 1486 RSDHLTA 1572 300 841 GAGTGTGAGG 1315 RSDNLAR 1401 QSSHLAS 1487 RSDNLAR 1573 300 770 TTCGCAGCCT 1316 DRSSLTR 1402 QSGSLTR 1488 RSDSLTK 1574 325 767 GGGGGAGCTG 1317 QSSDLAR 1403 QSGHLQR 1489 RSDHLSR 1575 335 800 TTGGCAGCCT 1318 ERGTLAR 1404 QSGSLTR 1490 RSDSLTK 1576 400 832 GACTAGGACC 1319 EKANLTR 1405 RSDNLTT 1491 DRSNLTR 1577 408 844 GAGATGGATC 1320 QSSNLQR 1406 RSDALRQ 1492 RSDNLQR 1578 444 683 GCTGCAGGAG 1321 QSGHLAR 1407 QSGSLTR 1493 QSSDLSR 1579 500 805 GCAGCGGTAG 1322 QRSALAR 1408 RSDELQR 1494 QSGSLTR 1580 500 839 GAGTGTGAGG 1323 RSDNLAR 1409 TSDHLAS 1495 RSDNLAR 1581 625 840 GAGTGTGAGG 1324 RSDNLAR 1410 MSHHLKT 1496 RSDNLAR 1582 625 830 GGAGAGTCGG 1325 RSDELRT 1411 RSDNLAR 1497 QPAHLAR 1583 683 831 GGAGAGTCGG 1326 RSDDLTK 1412 RSDNLAR 1498 QRAHLAR 1584 700 684 GCTGCAGGAG 1327 RSAHLAR 1413 QSGSLTR 1499 QSSDLSR 1585 850 846 GAGATGGATC 1328 QSSNLQR 1414 RRDVLNN 1500 RSDNLQR 1586 889.5 819 AAGTAGGGTG 1329 QSSHLTR 1415 RSDNLTT 1501 RSDNLTQ 1587 1000 820 ACGGTAGTTA 1330 QSSALTR 1416 QRSALAR 1502 RSDTLTQ 1588 1000 821 ACGGTAGTTA 1331 NRATLAR 1417 QRSALAR 1503 RSDTLTQ 1589 1000 822 GTGTGCTGGT 1332 RSDHLTT 1418 ERQHLAT 1504 RSDALAR 1590 1000 823 GTGTGCTGGT 1333 RSDHLTK 1419 ERQHLAT 1505 RSDALAR 1591 1000 824 GTGTGCTGGT 1334 RSDHLTT 1420 DRSHLRT 1506 RSDALAR 1592 1000 885 GTGTGCTGGT 1335 RSDHLTK 1421 DRSHLRT 1507 RSDALAR 1593 1000 886 TCAGTGGTAT 1336 QSSSLVR 1422 RSDALSR 1508 QSGDLRT 1594 1000 889 ATGGATTCAG 1337 QSGSLTT 1423 QQSNLVR 1509 RSDALTQ 1595 1000 890 CTGGTATGTC 1338 QRSHLTT 1424 QRSALAR 1510 RSDALRE 1596 1000 896 AAGTAGGGTG 1339 TSGHLVR 1425 RSDNLTT 1511 RSDNLTQ 1597 1000 898 ACGGTAGTTA 1340 NPATLAR 1426 QSSSLVR 1512 RSDTLTQ 1598 1000 899 CTGGTATGTC 1341 QRSHLTT 1427 QSSSLVR 1513 RSDALRE 1599 1000 900 CTGGTATGTC 1342 MSHHLKE 1428 QSSSLVR 1514 RSDALRE 1600 1000 901 CTGGTATGTC 1343 NSHHLKE 1429 QRSALAR 1515 RSDALRE 1601 1000 773 GCAGCGGTAG 1344 QSGALTR 1430 RSDELQR 1516 QSGSLTR 1602 1250 768 GGGGGAGCTG 1345 QSSDLAR 1431 QRAHLER 1517 RSDHLSR 1603 2000 681 GCTGCAGGAG 1346 RSAHLAR 1432 QSGDLTR 1518 QSSDLSR 1604 3000

TABLE 4 SEQ SEQ SEQ SEQ Kd SBS# TARGET ID F1 ID F2 ID F3 ID (nM) 607 AAGGTGGCAG 1605 QSGDLTR 1707 RSDSLAR 1809 RLDNRTA 1911 6.5 608 TTGGCTGGGC 1606 GSWHLTR 1708 QSSDLQR 1810 RSDSLTK 1912 8 611 GTGGCTGCAG 1607 QSGDLTR 1709 QSSDLQR 1811 RSDALAR 1913 11.5 612 GTGGCTGCAG 1608 QSGTLTR 1710 QSSDLQR 1812 RSDALAR 1914 0.38 613 TTGGCTGGGC 1609 RSDHLAR 1711 QSSDLQR 1813 RGDALTS 1915 1.45 614 TTGGCTGGGC 1610 RSDHLAR 1712 QSSDLQR 1814 RSDSLTK 1916 2 616 GAGGAGGATG 1611 QSSNLQR 1713 RSDNLAR 1815 RSDNLQR 1917 0.08 617 AAGGGGGGG 1612 RSDHLSR 1714 RSDHLTR 1816 RKDNMTA 1918 1 618 AAGGGGGGG 1613 RSDHLSR 1715 RSDHLTR 1817 RKDNNTQ 1919 0.55 619 AAGGGGGGG 1614 RSDHLSR 1716 RSDHLTR 1818 RKDNMTN 1920 1.34 620 AAGGGGGGG 1615 RSDHLSR 1717 RSDHLTR 1819 RLDNRTA 1921 0.54 621 AAGGGGGGG 1616 RSDHLSR 1718 RSDHLTR 1820 RLDNRTQ 1922 0.75 624 ACGGATGTCT 1617 DRSALAR 1719 TSANLAR 1821 RSDTLRS 1923 7 628 TTGTAGGGGA 1618 RSDHLTR 1720 RSDNLTT 1822 RGDALTS 1924 130 629 TTGTAGGGGA 1619 RSSHLTR 1721 RSDNLTT 1823 RGDALTS 1925 150 630 CGGGGAGAGT 1620 RSDNLAR 1722 QSGHLQR 1824 RSDHLRE 1926 37.5 646 TTGGTGGAAG 1621 QSGNLAR 1723 RSDALAR 1825 RGDALTS 1927 35 647 TTGGTGGAAG 1622 QSANLAR 1724 RSDALAR 1826 RGDALTS 1928 40 651 GTTGTGGAAT 1623 QSGNLSR 1725 RSDALAR 1827 NRATLAR 1929 67.5 652 TAGGAGGCTG 1624 QSSDLQR 1726 RSDNLAR 1828 RSDNLTT 1930 1.5 653 TAGGAGGCTG 1625 TTSDLTR 1727 RSDNLAR 1829 RSDNLTT 1931 5.5 654 TAGGCATAAA 1626 QSGNLRT 1728 QSGSLTR 1830 RSDNLTT 1932 105 655 TAGGCATAAA 1627 QSGNLRT 1729 QSSTLRR 1831 RSDNLTT 1933 1000 656 TAGGCATAAA 1628 QSGNLRT 1730 QSGSLTR 1832 RSDNLTS 1934 540 657 TAGGCATAAA 1629 QSGNLRT 1731 QSSTLRR 1833 RSDNLTS 1935 300 660 GAGGGAGTTC 1630 NRATLAR 1732 QSGHLTR 1834 RSDNLAR 1936 8.25 661 GAGGGAGTTC 1631 TTSALTR 1733 QSGHLTR 1835 RSDNLAR 1937 1.73 665 GCGGAGGCGC 1632 RSDDVTR 1734 RSDNLTR 1836 RSDDLRR 1938 12.5 689 AAGGCGGAGA 1633 RSDNLTR 1735 RSDELQR 1837 RLDNRTA 1939 82.5 692 AAGGCGGAGA 1634 RSDNLTR 1736 RSDELQR 1838 RSDNLTQ 1940 51 693 AAGGCGGAGA 1635 RSDNLTR 1737 RADTLRR 1839 RLDNRTA 1941 95 694 AAGGCGGAGA 1636 RSDNLTR 1738 EADTLRR 1840 RSDNLTQ 1942 28.5 695 GGGGGCGAGC 1637 RSSNLTR 1739 DRSHLAR 1841 RSDHLTR 1943 850 697 TGAGCGGCGG 1638 RSDELTR 1740 RSDELSR 1842 QSGHLTK 1944 200 698 TGAGCGGCGG 1639 RSDELTR 1741 RSDELSR 1843 QSHGLTS 1945 300 699 GCGGCGGCAG 1640 QSGSLTR 1742 RSDDLQR 1844 RSDERKR 1946 21.5 700 GCGGCGGCAG 1641 QSGDLTR 1743 RSDDLQR 1845 RSDERKR 1947 45 701 GCAGCGGAGC 1642 RSDNLAR 1744 RSDELQR 1846 QSGSLTR 1948 50.5 702 GCAGCGGAGC 1643 RSDNLAR 1745 RSDELQR 1847 QSGDLTR 1949 73.5 704 AAGGTGGCAG 1644 QSGDLTR 1746 RSDSLAR 1848 RSDNLTQ 1950 5 705 GGGGTGGGGC 1645 RSDHLAR 1747 RSDSLAR 1849 RSDHLSR 1951 0.01 706 GGGGTGGGGC 1646 RSDHLAR 1748 RSDSLLR 1850 RSDHLSR 1952 0.05 708 GAGTCGGAA 1647 QSANLAR 1749 RQDTLVG 1851 RSDNLAR 1953 300 709 GAGTCGGAA 1648 QSANLAR 1750 RKDVLVS 1852 RSDNLAR 1954 400 710 GAGTCGGAA 1649 QSGNLAR 1751 RLDGLRT 1853 RSDNLAR 1955 400 711 GAGTCGGAA 1650 QSGNLAR 1752 RQDTLVG 1854 RSDNLAR 1956 400 712 GGTGAGGAGT 1651 RSDNLAR 1753 RSDNLAR 1855 MSDHLSR 1957 9.5 713 GGTGAGGAGT 1652 RSDNLAR 1754 RSDNLAR 1856 MSHHLSR 1958 0.15 714 TGGGTCGCGG 1653 RSDELRR 1755 DRSALAR 1857 RSDHLTT 1959 200 715 TGGGTCGCGG 1654 RADTLRR 1756 DRSALAR 1858 RSDHLTT 1960 0.46 716 TTGGGAGCAC 1655 QSGSLTR 1757 QSGHLQR 1859 RGDALTS 1961 200 717 TTGGGAGCAC 1656 QSGSLTR 1758 QSGHLQR 1860 RSDALTK 1962 150 718 TTGGGAGCAC 1657 QSGSLTR 1759 QSGHLQR 1861 RSDALTR 1963 107.5 719 GGCATGGTGG 1658 RSDALTR 1760 RSDALTS 1862 DRSHLAR 1964 20 720 GAAGAGGATG 1659 TTSNLAR 1761 RSDNLAR 1863 QSGNLTR 1965 1.6 722 ATGGGGGTGG 1660 RSDALTR 1762 RSDHLTR 1864 RSDALRQ 1966 0.7 724 GGCATGGTGG 1661 RSDALTR 1763 RSDALRQ 1865 DRSHLAR 1967 2.5 725 GCTTGAGTTA 1662 QSSALAR 1764 QSGHLQK 1866 QSSDLQR 1968 3000 726 GAAGAGGATG 1663 QSSNLAR 1765 RSDNLAR 1867 QSGNLTR 1969 1.5 727 GCGGTGGCTC 1664 QSSDLTR 1766 RSDALSR 1868 RSDTLKK 1970 0.1 728 GGTGAGGAGT 1665 RSDNLAR 1767 RSDNLAR 1869 DSSKLSR 1971 15 729 GGAGGGGAGT 1666 RSDNLAR 1768 RSDHLSR 1870 QSGHLAR 1972 1000 730 TGGGTCGCGG 1667 RSDDLTR 1769 DRSALAR 1871 RSDHLTT 1973 1000 731 GTGGGGGAGA 1668 RSDNLAR 1770 RSDHLSR 1872 RSDALAR 1974 12 732 GCGGGTGGGG 1669 RSDHLAR 1771 QSSHLAR 1873 RSDDLTR 1975 22.5 733 GCGGGTGGGG 1670 RSDHLAR 1772 QSSHLAR 1874 RSDTLKK 1976 0.32 734 GGGGCTGGGT 1671 RSDHLAR 1773 QSSDLSR 1875 RSDHLSR 1977 0.25 735 GCGGTGGCTC 1672 QSSDLTR 1774 RSDALSR 1876 RSDERKR 1978 0.05 736 GAGGTGGGGA 1673 RSDHLAR 1775 RSDALSR 1877 RSDNLSR 1979 0.47 737 GGAGGGGAGT 1674 RSDNLAR 1776 RSDHLSR 1878 QRGHLSR 1980 1000 740 AAGGTGGCAG 1675 QSGSLTR 1777 RSDALAR 1879 RSDNRTA 1981 12.5 741 AAGGCTGAGA 1676 RSDNLTR 1778 QSSDLQR 1880 RSDNLTQ 1982 15 742 ACGGGGTTAT 1677 QRGALAS 1779 RSDHLSR 1881 RSDTLKQ 1983 29 743 ACGGGGTTAT 1678 QRGALAS 1780 RSDHLSR 1882 RSDTLTQ 1984 10 744 ACGGGGTTAT 1679 QRSALAS 1781 RSDHLSR 1883 RSDTLKQ 1985 8.33 745 ACGGGGTTAT 1680 QRSALAS 1782 RSDHLSR 1884 RSDTLTQ 1986 12.5 746 CTGGAAGCAT 1681 QSGSLTR 1783 QSGNLAR 1885 RSDALRE 1987 2.07 747 CTATTTTGGG 1682 RSDHLTT 1784 QSSALRT 1886 QSGALRE 1988 2000 748 TTGGACGGCG 1683 DSGHLTR 1785 DRSNLER 1887 RGDALTS 1989 112.3 749 TTGGACGGCG 1684 DRSHLTR 1786 DSSNLTR 1888 RGDALTS 1990 11.33 750 GAGGGAGCGA 1685 RSDELTR 1787 QSAHLAR 1889 RSDNLAR 1991 52 751 GGTGAGGAGT 1686 RSDNLAR 1788 RSDNLAR 1890 NRSHLAR 1992 7 752 GAGGTGGGGA 1687 RSHHLAR 1789 RSDALSR 1891 RSDNLSR 1993 31 757 CGGGCGGCTG 1688 QSSDLRR 1790 RSDELQR 1892 RSDHLRE 1994 14.5 758 CGGGCGGCTG 1689 QSSDLRR 1791 RADTLRR 1893 RSDHLRE 1995 16.5 759 TTGGACGGCG 1690 DSGHLTR 1792 DSSNLTR 1894 RGDALTS 1996 37 760 TTGGACGGCG 1691 DRSHLTR 1793 DRSNLER 1895 RGDALTS 1997 148.5 761 GCGGTGGCTC 1692 QSSDLQR 1794 RSDALSR 1896 RSDERKR 1998 6 762 GCGGTGGCTC 1693 QSSDLQR 1795 RSDALSR 1897 RSDTLKK 1999 18 776 ATGGACGGGT 1694 RSDHLAR 1796 DRSNLER 1898 RSDSLNQ 2000 0.4 777 ATGGACGGGT 1695 RSDHLAR 1797 DRSNLTR 1899 RSDALSA 2001 3.4 779 CGGGGAGCAG 1696 QSGSLTR 1798 QSGHLTR 1900 RSDHLAE 2002 0.5 780 CGGGGAGCAG 1697 QSGSLTR 1799 QSGHLTR 1901 RSDHLRA 2003 0.5 781 GGGGAGCAGC 1698 RSSNLRE 1800 RSDNLAR 1902 RSDHLTR 2004 4.25 783 TTGGGAGCGG 1699 RSDELTR 1801 QSGHLQR 1903 RGDALTS 2005 2000 785 TTGGGAGCGG 1700 RSDTLKK 1802 QSGHLQR 1904 RSDALTS 2006 50 786 TTGGGAGCGG 1701 RSDTLKK 1803 QSGHLQR 1905 RGDALRS 2007 2000 787 AGGGAGGATG 1702 QSDNLAR 1804 RSDNLAR 1906 RSDHLTQ 2008 4 826 GAGGGAGCGA 1703 RSDELTR 1805 QSGHLAR 1907 RSDNLAR 2009 2.75 827 GAGGGAGCGA 1704 RADTLRR 1806 QSGHLAR 1908 RSDNLAR 2010 1.2 882 GCGTGGGCGT 1705 RSDELTR 1807 RSDHLTT 1909 RSDERKR 2011 0.01 883 CCGTGGGCGT 1706 RSDELTR 1808 RSDHLTT 1910 RSDERKR 2012 1

TABLE 5 SEQ SEQ SEQ SEQ Kd SBS# TARGET ID F1 ID F2 ID F3 ID (Nm) 903 ATGGAAGGG 2013 RSDHLAR 2513 QSGNLAR 3013 RSDALRQ 3513 1.027 904 AAGGGTGAC 2014 DSSNLTR 2514 QSSHLAR 3014 RSDNLTQ 3514 1 905 GTGGTGGTG 2015 RSSALTR 2515 RSDSLAR 3015 RSDSLAR 3515 1.15 908 AAGGTCTCA 2016 QSGDLRT 2516 DRSALAR 3016 RSDNLRQ 3516 50 909 GTGGAAGAA 2017 QSGNLSR 2517 QSGNLQR 3017 RSDALAR 3517 16.4 910 ATGGAAGAT 2018 QSSNLAR 2518 QSGNLQR 3018 RSDALAQ 3518 0.03 911 ATGGGTGCA 2019 QSGSLTR 2519 QSSHLAR 3019 RSDALAQ 3519 0.91 912 TCAGAGGTG 2020 RSDSLAR 2520 RSDNLTR 3020 QSGDLRT 3520 0.135 914 CAGGAAAAG 2021 RSDNLTQ 2521 QSGNLAR 3021 RSDNLRE 3521 1.26 915 CAGGAAAAG 2022 RSDNLRQ 2522 QSGNLAR 3022 RSDNLRE 3522 45.15 916 GAGGAAGGA 2023 QSGHLAR 2523 QSGNLAR 3023 RSDNLQR 3523 1.3 919 TCATAGTAG 2024 RSDNLTT 2524 RSDNLRT 3024 QSGDLRT 3524 250 920 GATGTGGTA 2025 QSSSLVR 2525 RSDSLAR 3025 TSANLSR 3525 4 921 AAGGTCTCA 2026 QSGDLRT 2526 DPGALVR 3026 RSDNLRQ 3526 11 922 AAGGTCTCA 2027 QSHDLTK 2527 DRSALAR 3027 RSDNLRQ 3527 4 923 AAGGTCTCA 2028 QSHDLTK 2528 DPGALVR 3028 RSDNLRQ 3528 2 926 GTGGTGGTG 2029 RSDALTR 2529 RSDSLAR 3029 RSDSLAR 3529 7.502 927 CAGGTTGAG 2030 RSDNLAR 2530 TSGSLTR 3030 RSDNLRE 3530 3.61 928 CAGGTTGAG 2031 RSDNLAR 2531 QSSALTR 3031 RSDNLRE 3531 25 929 CAGGTAGAT 2032 QSSNLAR 2532 QSATLAR 3032 RSDNLRE 3532 1.3 931 GAGGAAGAG 2033 RSDNLAR 2533 QSSNLVR 3033 RSDNLAR 3533 2 932 ATGGAAGGG 2034 RSDHLAR 2534 QSSNLVR 3034 RSDALRQ 3534 797 933 GACGAGGAA 2035 QSANLAR 2535 RSDNLAR 3035 DRSNLTR 3535 500 934 ATGGAAGAT 2036 QSSNLAR 2536 QSGNLQR 3036 RSDALTS 3536 0.07 935 ATGGGTGCA 2037 QSGSLTR 2537 QSSHLAR 3037 RSDALTS 3537 0.91 937 GTGGGGGCT 2038 QSSDLTR 2538 RSDHLTR 3038 RSDSLAR 3538 0.03 938 GTGGGGGCT 2039 QSSDLRR 2539 RSDHLTR 3039 RSDSLAR 3539 0.049 939 GGGGGCTGG 2040 RSDHLTT 2540 DRSHLAR 3040 RSDHLSK 3540 0.352 940 GGGGGCTGG 2041 RSDHLTK 2541 DRSHLAR 3041 RSDHLSK 3541 1.5 941 GGGGCTGGG 2042 RSDHLAR 2542 QSSDLRR 3042 RSDKLSR 3542 0.077 942 GGGGCTGGG 2043 RSDHLAR 2543 QSSDLRR 3043 RSDHLSK 3543 0.13 943 GGGGCTGGG 2044 RSDHLAR 2544 TSGELVR 3044 RSDKLSR 3544 0.067 944 GGGGCTGGG 2045 RSDHLAR 2545 TSGELVR 3045 RSDHLSK 3545 0.027 945 GGTGCGGTG 2046 RSDSLTR 2546 RADTLRR 3046 MSHHLSR 3546 0.027 946 GGTGCGGTG 2047 RSDSLTR 2547 RSDVLQR 3047 MSHHLSR 3547 0.027 947 GGTGCGGTG 2048 RSDSLTR 2548 RSDELQR 3048 QSSHLAR 3548 0.013 948 GGTGCGGTG 2049 RSDSLTR 2549 RSDVLQR 3049 QSSHLAR 3549 0.017 962 GAGGCGGCA 2050 QSGSLTR 2550 RSDELQR 3050 RSDNLAR 3550 0.015 963 GAGGCGGCA 2051 QSGSLTR 2551 RSDDLQR 3051 RSDNLAR 3551 0.015 964 GCGGCGGTG 2052 RSDALAR 2552 RSDELQR 3052 RSDERKR 3552 0.041 965 GCGGCGGCC 2053 ERGDLTR 2553 RSDELQR 3053 RSDERKR 3553 3.1 966 GAGGAGGCC 2054 ERGTLAR 2554 RSDNLSR 3054 RSDNLAR 3554 0.028 967 GAGGAGGCC 2055 DRSSLTR 2555 RSDNLSR 3055 RSDNLAR 3555 0.055 968 GAGGCCGCA 2056 QSGSLTR 2556 DRSSLTR 3056 RSDNLAR 3556 1.4 969 GAGGCCGCA 2057 QSGSLTR 2557 DRSDLTR 3057 RSDNLAR 3557 0.275 970 GTGGGCGCC 2058 ERGTLAR 2558 DRSHLAR 3058 RSDALAR 3558 1.859 971 GTGGGCGCC 2059 DRSSLTR 2559 DRSHLAR 3059 RSDALAR 3559 0.144 972 GTGGGCGCC 2060 ERGDLTR 2560 DRSHLAR 3060 RSDALAR 3560 1.748 973 GCCGCGGTC 2061 DRSALTR 2561 RSDELQR 3061 ERGTLAR 3561 0.6 974 GCCGCGGTC 2062 DRSALTR 2562 RSDELQR 3062 DRSDLTR 3562 0.038 975 CAGGCCGCT 2063 QSSDLTR 2563 DRSSLTR 3063 RSDNLRE 3563 1.1 976 CAGGCCGCT 2064 QSSDLTR 2564 DRSDLTR 3064 RSDNLRE 3564 4.12 977 CTGGCAGTG 2065 RSDSLTR 2565 QSGSLTR 3065 RSDALRE 3565 0.017 978 CTGGCAGTG 2066 RSDSLTR 2566 QSGDLTR 3066 RSDALRE 3566 1.576 979 CTGGCGGCG 2067 RSSDLTR 2567 RSDELQR 3067 RSDALRE 3567 1.59 980 CTGGCGGCG 2068 RSDDLTR 2568 RSDELQR 3068 RSDALRE 3568 2.2 981 CAGGCGGCG 2069 RSDDLTR 2569 RSDELQR 3069 RSDNLRE 3569 0.375 982 CCGGGCTGG 2070 RSDHLTT 2570 DRSHLAR 3070 RSDELRE 3570 0.03 983 CCGGGCTGG 2071 RSDHLTK 2571 DRSHLAR 3071 RSDELRE 3571 1.385 984 GACGGCGAG 2072 RSDNLAR 2572 DRSHLAR 3072 DRSNLTR 3572 1.6 985 GACGGCGAG 2073 RSDNLAR 2573 DRSHLAR 3073 EKANLTR 3573 0.965 986 GGTGCTGAT 2074 QSSNLQR 2574 QSSDLQR 3074 MSHHLSR 3574 1.6 987 GGTGCTGAT 2075 QSSNLQR 2575 QSSDLQR 3075 TSGHLVR 3575 33.55 988 GGTGCTGAT 2076 TSGNLVR 2576 QSSDLQR 3076 MSHHLSR 3576 0.15 989 GGTGAGGGG 2077 RSDHLAR 2577 RSDNLAR 3077 MSHHLSR 3577 1.9 990 AAGGTGGGC 2078 DRSHLTR 2578 RSDSLAR 3078 RSDNLTQ 3578 5.35 991 AAGGTGGGC 2079 DRSHLTR 2579 SSGSLVR 3079 RSDNLTQ 3579 0.06 993 GGGGCTGGG 2080 RSDHLAR 2580 TSGELVR 3080 RSDHLSR 3580 3.1 994 GGGGGCTGG 2081 RSDHLTK 2581 DRSHLAR 3081 RSDHLSR 3581 0.03 995 GGGGAGGAA 2082 QSANLAR 2582 RSDNLAR 3082 RSDHLSK 3582 0.08 996 CAGTTGGTC 2083 DRSALAR 2583 RSDALTS 3083 RSDNLRE 3583 9.6 997 AGAGAGGCT 2084 QSSDLTR 2584 RSDNLAR 3084 QSGHLNQ 3584 1.65 998 ACGTAGTAG 2085 RSANLRT 2585 RSDNLTK 3085 RSDTLKQ 3585 0.23 999 AGAGAGGCT 2086 QSSDLTR 2586 RSDNLAR 3086 QSGKLTQ 3586 0.6 1000 CAGTTGGTC 2087 DRSALAR 2587 RSDALTR 3087 RSDNLRE 3587 11.15 1001 GGAGCTGAC 2088 EKANLTR 2588 QSSDLSR 3088 QPAHLAR 3588 1.8 1002 GCGGAGGAG 2089 RSDNLVR 2589 RSDNLAR 3089 RSDERKR 3589 0.028 1003 ACGTAGTAG 2090 RSANLRT 2590 RSDNLTK 3090 RSDTLRS 3590 0.118 1004 ACGTAGTAG 2091 RSDNLTT 2591 RSDNLTK 3091 RSDTLRS 3591 1.4 1006 GTAGGGGCG 2092 RSDDLTR 2592 RSDHLTR 3092 QRASLTR 3592 0.898 1007 GAGAGAGAT 2093 QSSNLQR 2593 QSGHLTR 3093 RLHNLAR 3593 167 1008 GAGATGGAG 2094 RSDNLSR 2594 RSDSLTQ 3094 RLHNLAR 3594 0.4 1009 GAGATGGAG 2095 RSDNLSR 2595 RSDSLTQ 3095 RSDNLSR 3595 1.9 1010 GAGAGAGAT 2096 QSSNLQR 2596 QSGHLTR 3096 RSDNLAR 3596 8.2 1011 TTGGTGGCG 2097 RSADLTR 2597 RSDSLAR 3097 RSDSLTK 3597 0.03 1012 GACGTAGGG 2098 RSDHLTR 2598 QSSSLVR 3098 DRSNLTR 3598 0.032 1013 GAGAGAGAT 2099 QSSNLQR 2599 QSGHLNQ 3099 RSDNLAR 3599 0.15 1014 GACGTAGGG 2100 RSDHLTR 2600 QSGSLTR 3100 DRSNLTR 3600 0.01 1015 GCGGAGGAG 2101 RSDNLVR 2601 RSDNLAR 3101 RSDTLKK 3601 0.008 1016 CAGTTGGTC 2102 DRSALAR 2602 RSDSLTK 3102 RSDNLRE 3602 0.09 1017 CTGGATGAC 2103 EKANLTR 2603 TSGNLVR 3103 RSDALRE 3603 0.233 1018 GTAGTAGAA 2104 QSANLAR 2604 QSSSLVR 3104 QPASLAR 3604 7.2 1019 AGGGAGGAG 2105 RSDNLAR 2605 RSDNLAR 3105 RSDHLTQ 3605 0.022 1020 ACGTAGTAG 2106 RSDNLTT 2606 RSDNLTK 3106 RSDTLKQ 3606 0.69 1022 GAGGAGGTG 2107 RSDALAR 2607 RSDNLAR 3107 RSDNLAR 3607 0.01 1024 GGGGAGGAA 2108 QSANLAR 2608 RSDNLAR 3108 RSDHLSR 3608 0.08 1025 GAGGAGGTG 2109 QSSALTR 2609 QSSSLVR 3109 RSDTLTQ 3609 0.115 1026 GTGGCTTGT 2110 MSHHLKE 2610 QSSDLSR 3110 RSDALAR 3610 0.076 1027 GCGGCGGTG 2111 RSDALAR 2611 RSDELQR 3111 RSDELQR 3611 0.054 1032 GGTGCTGAT 2112 TSGNLVR 2612 QSSDLQR 3112 TSGHLVR 3612 0.52 1033 GTGTTCGTG 2113 RSDALAR 2613 DRSALTT 3113 RSDALAR 3613 685.2 1034 GTGTTCGTG 2114 RSDALAR 2614 DRSALTK 3114 RSDALAR 3614 14.55 1035 GTGTTCGTG 2115 RSDALAR 2615 DRSALRT 3115 RSDALAR 3615 56 1037 GTAGGGGCA 2116 QSGSLTR 2616 RSDHLSR 3116 QRASLAR 3616 0.05 1038 GTAGGGGCA 2117 QTGELRR 2617 RSDHLSR 3117 QPASLAR 3617 0.152 1039 GGGGCTGGG 2118 RSDHLSR 2618 TSGELVR 3118 RSDHLTR 3618 1.37 1040 GGGGCTGGG 2119 RSDHLSR 2619 QSSDLQR 3119 RSDHLSK 3619 0.05 1041 TCATAGTAG 2120 RSDNLTT 2620 RSDNLRT 3120 QSHDLTK 3620 2.06 1043 CAGGGAGAG 2121 RSDNLAR 2621 QSGHLTR 3121 RSDNLRE 3621 0.16 1044 CAGGGAGAG 2122 RSDNLAR 2622 QRAHLER 3122 RSDNLRE 3622 1.07 1045 GGGGCAGGA 2123 QSGHLAR 2623 QSGSLTR 3123 RSDHLSR 3623 0.15 1046 GGGGCAGGA 2124 QSGHLAR 2624 QSGDLRR 3124 RSDHLSR 3624 0.09 1047 GGGGCAGGA 2125 QRAHLER 2625 QSGSLTR 3125 RSDHLSR 3625 24.7 1048 CAGGCTGTA 2126 QSGALTR 2626 QSSDLQR 3126 RSDNLRE 3626 1.387 1049 CAGGCTGTA 2127 QRASLAR 2627 QSSDLQR 3127 RSDNLRE 3627 55.6 1050 CAGGCTGTA 2128 QSSSLVR 2628 QSSDLQR 3128 RSDNLRE 3628 0.125 1051 GAGGCTGAG 2129 RSDNLTR 2629 QSSDLQR 3129 RSDNLVR 3629 0.02 1052 TAGGACGGG 2130 RSDHLAR 2630 EKANLTR 3130 RSDNLTT 3630 0.28 1053 TAGGACGGG 2131 RSDHLAR 2631 DRSNLTR 3131 RSDNLTT 3631 0.025 1054 GCTGCAGGG 2132 RSDHLAR 2632 QSGSLTR 3132 QSSDLQR 3632 0.033 1055 GCTGCAGGG 2133 RSDHLAR 2633 QSGSLTR 3133 TSGDLTR 3633 18.73 1056 GCTGCAGGG 2134 RSDHLAR 2634 QSGSLTR 3134 QSSDLQR 3634 0.045 1057 GCTGCAGGG 2135 RSDHLAR 2635 QSGDLTR 3135 TSGDLTR 3635 0.483 1058 GGGGCCGCG 2136 RSDELTR 2636 DRSSLTR 3136 RSDHLSR 3636 6.277 1059 GGGGCCGCG 2137 RSDELTR 2637 DRSDLTR 3137 RSDHLSR 3637 0.152 1060 GCGGAGGCC 2138 ERGTLAR 2638 RSDNLAR 3138 RSDERKR 3638 0.69 1061 GTTGCGGGG 2139 RSDHLAR 2639 RSDELQR 3139 QSSALTR 3639 0.165 1062 GTTGCGGGG 2140 RSDHLAR 2640 RSDELQR 3140 TSGSLTR 3640 0.068 1063 GTTGCGGGG 2141 RSDHLAR 2641 RSDELQR 3141 MSHALSR 3641 0.96 1064 GCGGCAGTG 2142 RSDALTR 2642 QSGSLTR 3142 RSDERKR 3642 0.453 1065 TGGGGCGGG 2143 RSDHLAR 2643 DRSHLAR 3143 RSDHLTT 3643 1.37 1066 GAGGGCGGT 2144 QSSHLTR 2644 DRSHLAR 3144 RSDNLVR 3644 0.15 1067 GAGGGCGGT 2145 TSGHLVR 2645 DRSHLAR 3145 RSDNLVR 3645 1.37 1068 GCAGGGGGC 2146 DRSHLTR 2646 RSDHLTR 3146 QSGDLTR 3646 2.05 1069 GCAGGCGGT 2147 DRSHLTR 2647 RSDHLTR 3147 QSGSLTR 3647 0.1 1070 GGGGCAGGC 2148 DRSHLTR 2648 QSGSLTR 3148 RSDHLSR 3648 0.456 1071 GGGGCAGGC 2149 DRSHLTR 2649 QSGDLTR 3149 RSDHLSR 3649 0.2 1072 GGATTGGCT 2150 QSSDLTR 2650 RSDALTT 3150 QRAHLAR 3650 0.46 1073 GGATTGGCT 2151 QSSDLTR 2651 RSDALTK 3151 QRAHLAR 3651 1.37 1075 GTGTTGGCG 2152 RSDELTR 2652 RSDALTK 3152 RSDALTR 3652 0.915 1076 GCGGCAGCG 2153 RSDELTR 2653 QSGSLTR 3153 RSDERKR 3653 4.1 1077 GCGGCAGCG 2154 RSDELTR 2654 QSGDLRR 3154 RSDERKR 3654 6.2 1078 GGGGGGGCC 2155 ERGTLAR 2655 RSDHLSR 3155 RSDHLSR 3655 0.2 1079 GGGGGGGCC 2156 ERGDLTR 2656 RSDHLSR 3156 RSDHLSR 3656 4.1 1080 CTGGAGGCG 2157 RSDELTR 2657 RSDNLAR 3157 RSDALRE 3657 1.37 1081 GGGGAGGTG 2158 RSDALTR 2658 RSDNLTR 3158 RSDHLSR 3658 0.05 1082 CTGGCGGCG 2159 RSDELTR 2659 RSDELTR 3159 RSDALRE 3659 0.152 1083 CTGGTGGCA 2160 QSGDLTR 2660 RSDALSR 3160 RSDALRE 3660 0.152 1084 GGTGAGGCG 2161 RSDELTR 2661 RSDNLAR 3161 MSHHLSR 3661 0.5 1085 GGTGAGGCG 2162 RSDELTR 2662 RSDNLAR 3162 QSSHLAR 3662 0.46 1086 GGGGCTGGG 2163 RSDHLSR 2663 QSSDLQR 3163 RSDHLTR 3663 0.1 1087 CGGGCGGCC 2164 ERGDLTR 2664 RSDELQR 3164 RSDHLAE 3664 1.24 1088 CGGGCGGCC 2165 ERGDLTR 2665 RSDELQR 3165 RSDHLRE 3665 0.905 1089 GACGAGGCT 2166 QSSDLRR 2666 RSDNLAR 3166 DRSNLTR 3666 0.171 1090 AAGGCGCTG 2167 RSDALRE 2667 RSDELQR 3167 RSDNLTQ 3667 30.3 1091 GTAGAGGAC 2168 DRSNLTR 2668 RSDNLAR 3168 QPASLAR 3668 0.085 1092 GCCTTGGCT 2169 QSSDLRR 2669 RGDALTS 3169 DRSDLTR 3669 2.735 1093 GCGGAGTCG 2170 RSADLRT 2670 RSDNLAR 3170 RSDERKR 3670 0.046 1094 GCGGTTGGT 2171 TSGHLVR 2671 QSSALTR 3171 RSDERKR 3671 12.34 1095 GGGGGAGCC 2172 ERGDLTR 2672 QRAHLER 3172 RSDHLSR 3672 0.395 1096 GGGGGAGCC 2173 DRSSLTR 2673 QRAHLER 3173 RSDHLSR 3673 0.019 1097 GAGGCCGAA 2174 QSANLAR 2674 DCRDLAR 3174 RSDNLAR 3674 0.77 1098 GCCGGGGAG 2175 RSDNLTR 2675 RSDHLTR 3175 DRSDLTR 3675 0.055 1099 GCGGAGTCG 2176 TSGHLVR 2676 TSGSLTR 3176 RSDERKR 3676 0.45 1100 GTGTTGGTA 2177 QSGALTR 2677 RGDALTS 3177 RSDALTR 3677 1.4 1101 ATGGGAGTT 2178 TTSALTR 2678 QRAHLER 3178 RSDALRQ 3678 0.065 1102 AAGGCAGAA 2179 QSANLAR 2679 QSGSLTR 3179 RSDNLTQ 3679 8.15 1103 AAGGCAGAA 2180 QSANLAR 2680 QSGDLTR 3180 RSDNLTQ 3680 1.4 1104 CGGGCAGCT 2181 QSSDLRR 2681 QSGSLTR 3181 RSDHLRE 3681 0.08 1105 CTGGCAGCC 2182 ERGDLTR 2682 QSGDLTR 3182 RSDALRE 3682 2.45 1106 CTGGCAGCC 2183 DRSSLTR 2683 QSGDLTR 3183 RSDALRE 3683 0.19 1107 GCGGGAGTT 2184 QSSALAR 2684 QRAHLER 3184 RSDERKR 3684 0.06 1108 CAGGCTGGA 2185 QSGHLAR 2685 TSGELVR 3185 RSDNLRE 3685 0.007 1109 AGGGGAGCC 2186 ERGDLTR 2686 QRAHLER 3186 RSDHLTQ 3686 0.347 1110 AGGGGAGCC 2187 DRSSLTR 2687 QRAHLER 3187 RSDHLTQ 3687 0.095 1111 CTGGTAGGG 2188 RSDHLAR 2688 QSSSLVR 3188 RSDALRE 3688 0.095 1112 CTGGTAGGG 2189 RSDHLAR 2689 QSATLAR 3189 RSDALRE 3689 0.125 1113 CTGGGGGCA 2190 QSGDLTR 2690 RSDHLTR 3190 RSDALRE 3690 0.06 1114 CAGGTTGAT 2191 QSSNLAR 2691 TSGSLTR 3191 RSDNLRE 3691 2.75 1115 CAGGTTGAT 2192 QSSNLAR 2692 QSSALTR 3192 RSDNLRE 3692 0.7 1116 CCGGAAGCG 2193 RSDELTR 2693 QSSNLVR 3193 RSDELRE 3693 12.3 1117 GCAGCGCAG 2194 RSSNLRE 2694 RSDELTR 3194 QSGSLTR 3694 2.85 1118 TAGGGAGTC 2195 DRSALTR 2695 QRAHLER 3195 RSDNLTT 3695 1.4 1119 TGGGAGGGT 2196 TSGHLVR 2696 RSDNLAR 3196 RSDHLTT 3696 0.1 1120 AGGGACGCG 2197 RSDELTR 2697 DRSNLTR 3197 RSDHLTQ 3697 2.735 1121 CTGGTGGCC 2198 ERGDLTR 2698 RSDALTR 3198 RSDALRE 3698 2.76 1122 CTGGTGGCC 2199 DRSSLTR 2699 RSDALTR 3199 RSDALRE 3699 0.101 1123 TAGGAAGCA 2200 QSGSLTR 2700 QSGNLAR 3200 RSDNLTT 3700 0.065 1124 GTGGATGGA 2201 QSGHLAR 2701 TSGNLVR 3201 RSDALTR 3701 0.101 1126 TTGGCTATG 2202 RSDALTS 2702 TSGELVR 3202 RGDALTS 3702 0.46 1127 CAGGGGGTT 2203 QSSALAR 2703 RSDHLTR 3203 RSDNLRE 3703 0.1 1128 AAGGTCGCC 2204 ERGDLTR 2704 DPGALVR 3204 RSDNLTQ 3704 5.45 1130 GGTGCAGAC 2205 DRSNLTR 2705 QSGDLTR 3205 MSHHLSR 3705 0.1 1131 GTGGGAGCC 2206 ERGDLTR 2706 QRAHLER 3206 RSDALTR 3706 0.95 1132 GGGGCTGGA 2207 QSCHLAR 2707 TSGELVR 3207 RSDHLSR 3707 0.055 1133 GGGGCTGGA 2208 QRAHLER 2708 TSGELVR 3208 RSDHLSR 3708 0.5 1134 TGGGGGTGG 2209 RSDHLTT 2709 RSDHLTR 3209 RSDHLTT 3709 0.067 1135 GCGGCGGGG 2210 RSDHLAR 2710 RSDELQR 3210 RSDERKR 3710 0.025 1136 CCGGGAGTG 2211 RSDALTR 2711 QRAHLER 3211 RSDTLRE 3711 0.225 1137 CCGGGAGTG 2212 RSSALTR 2712 QRAHLER 3212 RSDTLRE 3712 0.085 1138 CAGGGGGTA 2213 QSGALTR 2713 RSDHLTR 3213 RSDNLRE 3713 0.027 1139 ACGGCCGAG 2214 RSDNLAR 2714 DRSDLTR 3214 RSDTLTQ 3714 0.535 1140 AAGGGTGCG 2215 RSDELTR 2715 QSSHLAR 3215 RSDNLTQ 3715 0.3 1141 ATGGACTTG 2216 RGDALTS 2716 DRSNLTR 3216 RSDALTQ 3716 1.7 1148 TTGGAGGAG 2217 RSDNLTR 2717 RSDNLTR 3217 RGDALTS 3717 0.006 1149 TTGGAGGAG 2218 RSDNLTR 2718 RSDNLTR 3218 RSDALTK 3718 0.004 1150 GAAGAGGCA 2219 QSGSLTR 2719 RSDNLTR 3219 QSGNLTR 3719 0.004 1151 GTAGTATGG 2220 RSDHLTT 2720 QRSALAR 3220 QRASLAR 3720 1.63 1152 AAGGCTGGA 2221 QSGHLAR 2721 TSGELVR 3221 RSDNLTQ 3721 1.605 1153 AAGGCTGGA 2222 QRAHLAR 2722 TSGELVR 3222 RSDNLTQ 3722 8.2 1154 CTGGCGTAG 2223 RSDNLTT 2723 RSDELQR 3223 RSDALRE 3723 1.04 1156 ATGGTTGAA 2224 QSANLAR 2724 QSSALTR 3224 RSDALRQ 3724 7.2 1157 ATGGTTGAA 2225 QSANLAR 2725 TSGSLTR 3225 RSDALRQ 3725 0.885 1158 AGGGGAGAA 2226 QSANLAR 2726 QSGHLTR 3226 RSDHLTQ 3726 0.1 1159 AGGGGAGAA 2227 QSANLAR 2727 QRAHLER 3227 RSDHLTQ 3727 0.555 1160 TGGGAAGGC 2228 DRSHLAR 2728 QSSNLVR 3228 RSDHLTT 3728 0.415 1161 GAGGCCGCC 2229 DRSHLAR 2729 DRSDLTR 3229 RSDNLAR 3729 0.45 1162 GTGTTGGTA 2230 QSGALTR 2730 RADALMV 3230 RSDALTR 3730 0.465 1163 GTGTGAGCC 2231 ERGDLTR 2731 QSGHLTT 3231 RSDALTR 3731 1.45 1164 GTGTGAGCC 2232 ERGDLTR 2732 QSVHLQS 3232 RSDALTR 3732 15.4 1165 GCGAAGGTG 2233 RSDALTR 2733 RSDNLTQ 3233 RSDERKR 3733 1.4 1166 GCGAAGGTG 2234 RSDALTR 2734 RSDNLTQ 3234 RSSDRKR 3734 0.195 1167 GCGAAGGTG 2235 RSDALTR 2735 RSDNLTQ 3235 RSHDRKR 3735 0.95 1168 AAGGCGCTG 2236 RSDALRE 2736 RSSDLTR 3236 RSDNLTQ 3736 2.8 1169 GTAGAGGAC 2237 DRSNLTR 2737 RSDNLAR 3237 QSSSLVR 3737 0.053 1170 GCCTTGGCT 2238 QSSDLRR 2738 RADALMV 3238 DRSDLTR 3738 2.75 1171 GCGGAGTCG 2239 RSDDLRT 2739 RSDNLAR 3239 RSDERKR 3739 0.18 1172 GCCGGGGAG 2240 RSDNLTR 2740 RSDHLTR 3240 ERGDLTR 3740 0.01 1173 GCTGAAGGG 2241 RSDHLSR 2741 QSGNLAR 3241 QSSDLRR 3741 0.008 1174 GCTGAAGGG 2242 RSDHLSR 2742 QSSNLVR 3242 QSSDLRR 3742 0.018 1175 AAGGTCGCC 2243 DRSDLTR 2743 DPGALVR 3243 RSDNLTQ 3743 8.9 1176 GTGGGAGCC 2244 DRSDLTR 2744 QRAHLER 3244 RSDALTR 3744 4.1 1177 CCGGGCGCA 2245 QSGSLTR 2745 DRSHLAR 3245 RSDTLRE 3745 4.1 1178 GAGGATGGC 2246 DRSHLAR 2746 TSGNLVR 3246 RSDNLAR 3746 0.085 1179 GCAGCGCAG 2247 RSSNLRE 2747 RSSDLTR 3247 QSGSLTR 3747 2.735 1180 AAGGAAAGA 2248 QSGHLNQ 2748 QSGNLAR 3248 RSDNLTQ 3748 4.825 1181 TTGGCTATG 2249 RSDALRQ 2749 TSGELVR 3249 RGDALTS 3749 8.2 1182 CAGGAAGGC 2250 DRSHLAR 2750 QSGNLAR 3250 RSDNLRE 3750 1.48 1183 CAGGAAGGC 2251 DRSHLAR 2751 QSSNLVR 3251 RSDNLRE 3751 1.935 1184 AAGGAAAGA 2252 KNWKLQA 2752 QSGNLAR 3252 RSDNLTQ 3752 2.785 1185 AAGGAAAGA 2253 KNWKLQA 2753 QSHNLAR 3253 RSDNLTQ 3753 5.25 1186 GCCGAGGTG 2254 RSDSLLR 2754 RSKNLQR 3254 ERGTLAR 3754 27.5 1187 CTGGTGGGC 2255 DRSHLAR 2755 RSDALTR 3255 RSDALRE 3755 0.006 1188 GTAGTATGG 2256 RSDHLTT 2756 QSSSLVR 3256 QRASLAR 3756 2.74 1189 ATGGTTGAA 2257 QSANLAR 2757 TSGALTR 3257 RSDALRQ 3757 1.51 1190 ATGGCAGTG 2258 RSDALTR 2758 QSGDLTR 3258 RSDSLNQ 3758 1.484 1191 ATGGCAGTG 2259 RSDALTR 2759 QSGSLTR 3259 RSDSLNQ 3759 5.325 1192 ATGGCAGTG 2260 RSDALTR 2760 QSGDLTR 3260 RSDALTQ 3760 2.364 1193 ATGGCAGTG 2261 RSDALTR 2761 QSGSLTR 3261 RSDALTQ 3761 3.125 1194 GAGAAGGTG 2262 RSDALTR 2762 RSDNRTA 3262 RSDNLTR 3762 2.19 1195 GAGAAGGTG 2263 RSDALTR 2763 RSDNRTA 3263 RSSNLTR 3763 2.8 1197 GAAGGTGCC 2264 ERCDLTR 2764 MSHHLSR 3264 QSGNLTR 3764 14.8 1199 ATGGAGAAG 2265 RSDNRTA 2765 RSDNLTR 3265 RSDALTQ 3765 3.428 1200 ATGCAGAAG 2266 RSDNRTA 2766 RSSNLTR 3266 RSDALTQ 3766 16.87 1201 ATGGAGAAG 2267 RSDNRTA 2767 RSHNLTR 3267 RSDALTQ 3767 14.8 1202 CTGGAGTAC 2268 DRSNLRT 2768 RSDNLTR 3268 RSDALRE 3768 2.834 1203 GGAGTACTG 2269 RSDALRE 2769 QRSALAR 3269 QRAHLAR 3769 2.945 1204 GGAGTACTG 2270 RSDALRE 2770 QSSSLVR 3270 QRAHLAR 3770 4.38 1205 CGGGCAGCT 2271 QSSDLRR 2771 QSGDLTR 3271 RSDHLRE 3771 0.9 1206 GCGGGAGTT 2272 TTSALTR 2772 QRAHLER 3272 RSDERKR 3772 0.034 1207 CAGGCTGGA 2273 QRAHLER 2773 TSGELVR 3273 RSDNLRE 3773 0.45 1209 CCGGAAGCG 2274 RSDELTR 2774 QSSNLVR 3274 RSDTLRE 3774 19.28 1211 GCAGCGCAG 2275 RSDNLRE 2775 RSDELTR 3275 QSGSLTR 3775 6.5 1212 CAGGGGGTT 2276 TTSALTR 2776 RSDHLTR 3276 RSDNLRE 3776 0.05 1213 GAAGAAGAG 2277 RSDNLTR 2777 QSSNLVR 3277 QSGNLTR 3777 12.3 1214 ATGGGAGTT 2278 TTSALTR 2778 QRAHLER 3278 RSDALTQ 3778 0.46 1215 GTGGGGGCT 2279 QSSDLRR 2779 RSDHLTR 3279 RSDALTR 3779 0.003 1217 GAAGAGGCA 2280 QSGSLTR 2780 RSDNLTR 3280 QSANLTR 3780 0.004 1218 GCGGTGAGG 2281 RSDHLTQ 2781 RSQALTR 3281 RSDERKR 3781 0.46 1219 AAGGAAAGG 2282 RSDHLTQ 2782 QSHNLAR 3282 RSDNLTQ 3782 0.68 1220 AAGGAAAGG 2283 RSDHLTQ 2783 QSGNLAR 3283 RSDNLTQ 3783 0.175 1221 AAGGAAAGG 2284 RSDHLTQ 2784 QSSNLVR 3284 RSDNLTQ 3784 1.4 1222 CAGGAGGGC 2285 DRSHLAR 2785 RSDNLAR 3285 RSDNLRE 3785 0.155 1223 ATGGACTTG 2286 RSDALTK 2786 DRSNLTR 3286 RSDALTQ 3786 7 1224 ATGGACTTG 2287 RADALMV 2787 DRSNLTR 3287 RSDALTQ 3787 12 1227 GAATAGGGG 2288 RSDHLSR 2788 RSDHLTK 3288 QSGNLAR 3788 25 1228 ACGGCCGAG 2289 RSDNLAR 2789 DRSDLTR 3289 RSDDLTQ 3789 12 1229 AAGGGTGCG 2290 RSDELTR 2790 MSHHLSR 3290 RSDNLTQ 3790 8.2 1230 AAGGGAGAC 2291 DRSNLTR 2791 QSGHLTR 3291 RSDNLTQ 3791 0.383 1231 AAGGGAGAC 2292 DRSNLTR 2792 QRAHLER 3292 RSDNLTQ 3792 0.213 1232 TGGGACCTG 2293 RSDALRE 2793 DRSNLTR 3293 RSDHLTT 3793 0.113 1233 TGGGACCTG 2294 RSDALRE 2794 DRSNLTR 3294 RSDHLTT 3794 0.635 1234 GAGTAGGCA 2295 QSGSLTR 2795 RSDNLTK 3295 RSDNLAR 3795 0.101 1236 GAGTAGGCA 2296 QSGSLTR 2796 RSDHLTT 3296 RSDNLAR 3796 0.065 1237 GAAGGAGAG 2297 RSDNLAR 2797 QRAHLER 3297 QSGNLAR 3797 0.065 1238 CTGGATGTT 2298 QSSALAR 2798 TSGNLVR 3298 RSDALRE 3798 0.313 1239 CAGGACGTG 2299 RSDALTR 2799 DPGNLVR 3299 RSDNLKD 3799 0.144 1240 GGGGAGGCA 2300 QSGSLTR 2800 RSDNLTR 3300 RSDHLSR 3800 0.056 1241 GAGGTGTCA 2301 QSHDLTK 2801 RSDALAR 3301 RSDNLAR 3801 0.027 1242 GGGGTTGAA 2302 QSMJLAR 2802 TSGSLTR 3302 RSDHLSR 3802 0.02 1243 GGGGTTGAA 2303 QSANLAR 2803 QSSALTR 3303 RSDHLSR 3803 0.101 1244 GTCGCGGTG 2304 RSDALTR 2804 RSDELQR 3304 DRSALAR 3804 0.044 1245 GTCGCGGTG 2305 RSDALTR 2805 RSDELQR 3305 DSGSLTR 3805 0.102 1246 GTGGTTGCG 2306 RSDELTR 2806 TSGSLTR 3306 RSDALTR 3806 0.051 1247 GTGGTTGCG 2307 RSDELTR 2807 TSGALTR 3307 RSDALTR 3807 0.117 1248 GTCTAGGTA 2308 QSGALTR 2808 RSDNLTT 3308 DRSALAR 3808 5.14 1249 CCGGGAGCG 2309 RSDELTR 2809 QSGHLTR 3309 RSDTLRE 3809 0.26 1250 GAAGGAGAG 2310 RSDNLAR 2810 QSGHLTR 3310 QSGNLAR 3810 0.31 1252 CCGGCTGGA 2311 QRAHLER 2811 QSSDLTR 3311 RSDTLRE 3811 0.153 1253 CCGGGAGCG 2312 RSDELTR 2812 QRAHLER 3312 RSDTLRE 3812 0.228 1255 ACGTAGTAG 2313 RSDNLTT 2813 RSDNLTK 3313 RSDTLKQ 3813 0.69 1256 GGGGAGGAT 2314 QSSNLAR 2814 RSDNLQR 3314 RSDHLSR 3814 2 1257 GGGGAGGAT 2315 TTSNLAR 2815 RSDNLQR 3315 RSDHLSR 3815 1 1258 GGGGAGGAT 2316 QSSNLRR 2816 RSDNLQR 3316 RSDHLSR 3816 2 1259 GAGTGTGTG 2317 RSDSLLR 2817 DRDHLTR 3317 RSDNLAR 3817 1.5 1260 GAGTGTGTG 2318 RLDSLLR 2818 DRDHLTR 3318 RSDNLAR 3818 1.8 1261 TGCGGGGCA 2319 QSGDLTR 2819 RSDHLTR 3319 RRDTLHR 3819 0.2 1262 TGCGGGGCA 2320 QSGDLTR 2820 RSDHLTR 3320 RLDTLGR 3820 3 1263 TGCGGGGCA 2321 QSGDLTR 2821 RSDHLTR 3321 DSGHLAS 3821 21 1264 AAGTTGGTT 2322 TTSALTR 2822 RADALMV 3322 RSDNLTQ 3822 0.21 1265 AAGTTGGTT 2323 TTSALTR 2823 RSDALTT 3323 RSDNLTQ 3823 0.077 1266 CAGGGTGGC 2324 DRSHLTR 2824 QSSHLAR 3324 RSDNLRE 3824 6.1 1267 TAGGCAGTC 2325 DRSALTR 2825 QSGSLTR 3325 RSDNLTT 3825 6 1268 CTGTTGGCT 2326 QSSDLTR 2826 RADALMV 3326 RSDALRE 3826 1.52 1269 CTGTTGGCT 2327 QSSDLTR 2827 RSDALTT 3327 RSDALRE 3827 12.3 1270 TTGGATGGA 2328 QSGHLAR 2828 TSGNLVR 3328 RSDALTK 3828 0.4 1271 GTGGCACTG 2329 RSDALRE 2829 QSGSLTR 3329 RSDALTR 3829 0.915 1272 CAGGAGTCC 2330 DRSSLTT 2830 RSDNLAR 3330 RSDNLRE 3830 0.04 1273 CAGGAGTCC 2331 ERGDLTT 2831 RSDNLAR 3331 RSDNLRE 3831 0.1 1274 GCATGGGAA 2332 QSANLSR 2832 RSDHLTT 3332 QSGSLTR 3832 0.306 1275 GCATGGGAA 2333 QRSNLVR 2833 RSDHLTT 3333 QSGSLTR 3833 0.326 1276 TAGGAAGAG 2334 RSDNLAR 2834 QRSNLVR 3334 RSDNLTT 3834 0.685 1277 GAAGAGGGG 2335 RSDHLAR 2835 RSDNLAR 3335 QSGNLTR 3835 0.421 1278 GAGTAGGCA 2336 QSGSLTR 2836 RSDNLRT 3336 RSDNLAR 3836 0.019 1279 GAGGTGTCA 2337 QSGDLRT 2837 RSDALAR 3337 RSDNLAR 3837 0.025 1282 TCGGTCGCC 2338 ERGDLTR 2838 DPGALVR 3338 RSDELRT 3838 74.1 1287 GTGGTAGGA 2339 QSGHLAR 2839 QSGALAR 3339 RSDALTR 3839 0.152 1288 CAGGGTGGC 2340 DRSHLTR 2840 QSSHLAR 3340 RSDNLTE 3840 4.1 1289 TAGGCAGTC 2341 DRSALTR 2841 QSGSLTR 3341 RSDNLTK 3841 1.37 1290 GTGGTGATA 2342 QSGALTQ 2842 RSHALTR 3342 RSDALTR 3842 24.05 1291 GTGGTGATA 2343 QQASLNA 2843 RSHALTR 3343 RSDALTR 3843 20.55 1292 TTGGATGGA 2344 QSGHLAR 2844 TSGNLVR 3344 RSDALTT 3844 4.12 1293 AAGGTAGGT 2345 TSGHLVR 2845 QSGALAR 3345 RSDNLTQ 3845 0.457 1294 AAGGTAGGT 2346 MSHHLSR 2846 QSGALAR 3346 RSDNLTQ 3846 2.75 1295 CAGGAGTCC 2347 DRSSLTT 2847 RSDNLAR 3347 RSDNLTE 3847 0.116 1296 CAGGAGTCC 2348 ERGDLTT 2848 RSDNLAR 3348 RSDNLTE 3848 37 1297 TAGGAAGAG 2349 RSDNLAR 2849 QRSNLVR 3349 RSDNLTK 3849 0.05 1298 CAGGACGTG 2350 RSDLATR 2850 DPGNLVR 3350 RSDNLTE 3850 0.05 1300 GTCTAGGTA 2351 QSGALTR 2851 RSDNLTK 3351 DRSALAR 3851 0.46 1302 CCGGCTGGA 2352 QSGHLTR 2852 QSSDLTR 3352 RSDTLRE 3852 0.05 1303 TAGGAGTTT 2353 QRSALAS 2853 RSDNLAR 3353 RSDNLTT 3853 0.088 1306 CTGGCCTTG 2354 RSDALTT 2854 DCRDLAR 3354 RSDALRE 3854 2.285 1308 TGGGCAGCC 2355 ERGTLAR 2855 QSGSLTR 3355 RSDHLTT 3855 0.305 1309 TAGGAGTTT 2356 QSSALAS 2856 RSDNLAR 3356 RSDNLTT 3856 0.184 1310 TAGGAGTTT 2357 TTSALAS 2857 RSDNLAR 3357 RSDNLTT 3857 0.075 1311 TGGGCAGCC 2358 ERGDLAR 2858 QSGSLTR 3358 RSDHLTT 3858 0.91 1312 GGGGCGTGA 2359 QSGHLTK 2859 RSDELQR 3359 RSDHLSR 3859 0.23 1313 GGGGCGTGA 2360 QSCHLTT 2860 RSDELQR 3360 RSDHLSR 3860 0.09 1314 GTACAGTAG 2361 RSDNLTT 2861 RSDNLRE 3361 QSSSLVR 3861 3.09 1315 GTACAGTAG 2362 RSDNLTT 2862 RSDNLTE 3362 QSSSLVR 3862 9.27 1318 ATGGTGTGT 2363 TSSHLAS 2863 RSDALAR 3363 RSDALAQ 3863 0.048 1319 ATGGTGTGT 2364 MSHHLTT 2864 RSDALAR 3364 RSDALAQ 3864 0.228 1320 TTGGGAGAG 2365 RSDNLAR 2865 QPAHLER 3365 RSDALTT 3865 0.044 1321 TTGGGAGAG 2366 RSDNLAR 2866 QRAHLER 3366 RADALMV 3866 0.127 1322 GTGGGAATA 2367 QSGALTQ 2867 QSGHLTR 3367 RSDALTR 3867 0.799 1323 GTGGGAATA 2368 QLTGLNQ 2868 QSGHLTR 3368 RSDALTR 3868 0.744 1324 GTGGGAATA 2369 QQASLNA 2869 QSHHLTR 3369 RSDALTR 3869 18.52 1325 TTGGTTGGT 2370 TSGHLVR 2870 TSGSLTR 3370 RSDALTK 3870 0.306 1326 TTGGTTGGT 2371 TSGHLVR 2871 QSSALTR 3371 RSDALTK 3871 4.385 1327 TTGGTTGGT 2372 TSGHLVR 2872 TSGSLTR 3372 RSDALTT 3872 0.566 1328 TTGGTTGGT 2373 TSGHLVR 2873 QSSALTR 3373 RSDALTT 3873 7.95 1329 CTGGCCTGG 2374 RSDHLTT 2874 DRSDLTR 3374 RSDALRE 3874 0.68 1330 GAGGTGTGA 2375 QSGHLTT 2875 RSDALTR 3375 RSDNLAR 3875 0.175 1331 CTGGCCTGG 2376 RSDHLTT 2876 DCRDLAR 3376 RSDALRE 3876 0.388 1334 CCGGCGCTG 2377 RSDALRE 2877 RSSDLTR 3377 RSDDLRE 3877 0.31 1335 GACGCTGGC 2378 DRSHLTR 2878 QSSDLTR 3378 DSSNLTR 3878 1.4 1336 CGGGCTGGA 2379 QSGHLAR 2879 QSSDLTR 3379 RSDHLAE 3879 1.4 1337 CGGGCTGGA 2380 QSSHLAR 2880 QSSDLTR 3380 RSDHLAE 3880 0.235 1338 GGGATGGCG 2381 RSDELTR 2881 RSDALTQ 3381 RSDHLSR 3881 1.04 1339 GGGATGGCG 2382 RSDELTR 2882 RSDSLTQ 3382 RSDHLSR 3882 0.569 1340 GGGATGGCG 2383 RSDELTR 2883 RSDALTQ 3383 RSHHLSR 3883 0.751 1341 GGGATGGCG 2384 RSDELTR 2884 RSDSLTQ 3384 RSHHLSR 3884 4.1 1342 CAGGCGCAG 2385 RSDNLRE 2885 RSSDLTR 3385 RSDNLTE 3885 0.68 1343 CAGGCGCAG 2386 RSDNLTT 2886 RTSTLTR 3386 RSDNLTE 3886 37.04 1344 CCGGGCGAC 2387 DRSNLTR 2887 DRSHLAR 3387 RSDTLRE 3887 2.28 1346 GATGTGTGA 2388 QSGHLTT 2888 RSDALAR 3388 TSANLSR 3888 0.153 1347 CAGTGAATG 2389 RSDALTS 2889 QSHHLTT 3389 RSDNLTE 3889 8.23 1348 GGGTCACTG 2390 RSDALTA 2890 QAATLTT 3390 RSDHLSR 3890 2.58 1350 CAGTGAATG 2391 RSDALTQ 2891 QSGHLTT 3391 RSDNLTE 3891 74.1 1351 GGGTCACTG 2392 RSDALRE 2892 QSHDLTK 3392 RSDHLSR 3892 0.234 1352 GTGTGGGTC 2393 DRSALAR 2893 RSDHLTT 3393 RSDALTR 3893 0.023 1353 CTGGCGAGA 2394 QSGHLNQ 2894 RSDELQR 3394 RSDALRE 3894 56.53 1354 CTGGCGAGA 2395 KNWKLQA 2895 RSDELQR 3395 RSDALRE 3895 20.85 1355 GCTTTGGCA 2396 QSGSLTR 2896 RSDALTT 3396 QSSDLTR 3896 0.172 1356 GCTTTGGCA 2397 QSGSLTR 2897 RADALMV 3397 QSSDLTR 3897 0.034 1357 GACTTGGTA 2398 QSSSLVR 2898 RSDALTT 3398 DRSNLTR 3898 0.032 1358 GACTTGGTA 2399 QSSSLVR 2899 RADALMV 3399 DRSNLTR 3899 0.05 1360 CAGTTGTGA 2400 QSGHLTT 2900 RADALMV 3400 RSDNLTE 3900 41.7 1361 AAGGAAAAA 2401 QKTNLDT 2901 QSGNLQR 3401 RSDNLTQ 3901 0.835 1362 AAGGAAAAA 2402 QSGNLNQ 2902 QSGNLQR 3402 RSDNLTQ 3902 0.332 1363 AAGGAAAAA 2403 QKTNLDT 2903 QRSNLVR 3403 RSDNLTQ 3903 74.1 1364 ATGGGTGAA 2404 QSANLSR 2904 QSSHLAR 3404 RSDALAQ 3904 1.22 1365 ATGGGTGAA 2405 QRSNLVR 2905 QSSHLAR 3405 RSDALAQ 3905 0.152 1366 ATGGGTGAA 2406 QSANLSR 2906 TSGHLVR 3406 RSDALAQ 3906 22.63 1367 ATGGGTGAA 2407 QRSNLVR 2907 TSGHLVR 3407 RSDALAQ 3907 1.028 1368 CTGGGAGAT 2408 QSSNLAR 2908 QRAHLER 3408 RSDALRE 3908 0.051 1369 CTGGGAGAT 2409 QSSNLAR 2909 QSGHLTR 3409 RSDALRE 3909 0.227 1373 GTGGTGGGC 2410 DRSHLTR 2910 RSDALSR 3410 RSDALTR 3910 0.025 1374 CCGGCGGTG 2411 RSDALTR 2911 RSDELQR 3411 RSDELRE 3911 0.003 1375 CCGGCGGTG 2412 RSDALTR 2912 RSDDLQR 3412 RSDELRE 3912 0.008 1376 CCGGCGGTG 2413 RSDALTR 2913 RSDERKR 3413 RSDELRE 3913 0.858 1377 CCGGCGGTG 2414 RSDALTR 2914 RSDELQR 3414 RSDDLRE 3914 0.012 1378 CCGGCGGTG 2415 RSDALTR 2915 RSDDLQR 3415 RSDDLRE 3915 0.012 1379 CCGGCGGTG 2416 RSDALTR 2916 RSDERKR 3416 RSDDLRE 3916 0.25 1380 GCCGACGGT 2417 QSSHLTR 2917 DRSNLTR 3417 ERGDLTR 3917 0.076 1381 GCCGACGGT 2418 QSSHLTR 2918 DPGNLVR 3418 ERGDLTR 3918 0.23 1382 GCCGACGGT 2419 QSSHLTR 2919 DRSNLTR 3419 DCRDLAR 3919 3.1 1383 GCCGACGGT 2420 QSSHLTR 2920 DPGNLVR 3420 DGRDLAR 3920 1.74 1384 GGTGTGGGC 2421 DRSHLTR 2921 RSDALSR 3421 MSHHLSR 3921 0.013 1385 TGGGCAAGA 2422 QSGHLNQ 2922 QSGSLTR 3422 RSDHLTT 3922 0.229 1386 TGGGCAAGA 2423 ENWKLQA 2923 QSGSLTR 3423 RSDHLTT 3923 0.193 1389 CTGGCCTGG 2424 RSDHLTT 2924 DCRDLAR 3424 RSDALRE 3924 0.175 1393 TGGGAAGCT 2425 QSSDLRR 2925 QSGNLAR 3425 RSDHLTT 3925 0.1 1394 TGGGAAGCT 2426 QSSDLRR 2926 QSGNLAR 3426 RSDHLTK 3926 0.04 1395 GAAGAGGGA 2427 QSGHLQR 2927 RSDNLAR 3427 QSGNLAR 3927 0.025 1396 GAAGAGGGA 2428 QRAHLAR 2928 RSDNLAR 3428 QSGNLAR 3928 0.107 1397 GAAGAGGGA 2429 QSSHLAR 2929 RSDNLAR 3429 QSGNLAR 3929 0.14 1398 TAATGGGGG 2430 RSDHLSR 2930 RSDHLTT 3430 QSGNLRT 3930 0.065 1399 TGGGAGTGT 2431 TKQHLKT 2931 RSDNLAR 3431 RSDHLTT 3931 0.1 1400 CCGGGTGAG 2432 RSDNLAR 2932 QSSHLAR 3432 RSDDLRE 3932 0.371 1401 GAGTTGGCC 2433 ERGTLAR 2933 RADALMV 3433 RSDNLAR 3933 0.167 1402 CTGGAGTTG 2434 RGDALTS 2934 RSDNLAR 3434 RSDALRE 3934 0.15 1403 ATGGCAATG 2435 RSDALTQ 2935 QSGSLTR 3435 RSDALTQ 3935 0.07 1404 GAGGCAGGG 2436 RSDHLSR 2936 QSGSLTR 3436 RSDNLAR 3936 0.022 1405 GAGGCAGGG 2437 RSDHLSR 2937 QSGDLTR 3437 RSDNLAR 3937 0.045 1406 GAAGCGGAG 2438 RSDNLAR 2938 RSDELTR 3438 QSGNLAR 3938 0.025 1407 GCGGGCGCA 2439 QSGSLTR 2939 DRSHLAR 3439 RSDERKR 3939 0.585 1408 CCGGCAGGG 2440 RSDHLSR 2940 QSGSLTR 3440 RSDELRE 3940 0.305 1409 CCGGCAGGG 2441 RSDHLSR 2941 QSGSLTR 3441 RSDDLRE 3941 0.153 1410 CCGGCGGCG 2442 RSDELTR 2942 RSDELQR 3442 RSDELRE 3942 0.814 1411 TGAGGCGAG 2443 RSDNLAR 2943 DRSHLAR 3443 QSGHLTK 3943 0.282 1412 CTGGCCGTG 2444 RSDSLLR 2944 ERGTLAR 3444 RSDALRE 3944 0.172 1413 CTGGCCGCG 2445 RSDELTR 2945 DRSDLTR 3445 RSDALRE 3945 0.152 1414 CTGGCCGCG 2446 RSDELTR 2946 ERGTLAR 3446 RSDALRE 3946 0.914 1415 GCGGCCGAG 2447 RSDNLAR 2947 DRSDLTR 3447 RSDELQR 3947 0.102 1416 GCGGCCGAG 2448 RSDNLAR 2948 ERGTLAR 3448 RSDELQR 3948 0.153 1417 GAGTTGGCC 2449 ERGTLAR 2949 RGDALTS 3449 RSDNLAR 3949 1.397 1418 CTGGAGTTG 2450 RADALMV 2950 RSDNLAR 3450 RSDALRE 3950 0.241 1422 GGGTCGGCG 2451 RSDELTR 2951 RSDDLTT 3451 RSDHLSR 3951 0.064 1423 GGGTCGGCG 2452 RSDELTR 2952 RSDDLTK 3452 RSDHLSR 3952 0.034 1424 CAGGGCCCG 2453 RSDELRE 2953 DRSHLAR 3453 RSDNLRE 3953 1.37 1427 CAGGGCCCG 2454 RSDDLRE 2954 DRSHLAR 3454 RSDNLTE 3954 0.271 1428 TGAGGCGAG 2455 RSDNLAR 2955 DRSHLAR 3455 QSVHLQS 3955 0.102 1429 TGAGGCGAG 2456 RSDNLAR 2956 DRSHLAR 3456 QSGHLTT 3956 0.074 1430 TCGGCCGCC 2457 ERGTLAR 2957 DRSDLTR 3457 RSDDLTK 3957 0.352 1431 TCGGCCGCC 2458 ERGTLAR 2958 DRSDLTR 3458 RSDDLAS 3958 6.17 1432 TCGGCCGCC 2459 ERGTLAR 2959 ERGTLAR 3459 RSDDLTK 3959 1.778 1434 CTGGCCGTG 2460 RSDSLLR 2960 DRSDLTR 3460 RSDALRE 3960 0.051 1435 TAATGGGGG 2461 RSDHLSR 2961 RSDHLTT 3461 QSGNLTK 3961 0.057 1436 TGGGAGTGT 2462 TSDHLAS 2962 RSDNLAR 3462 RSDHLTT 3962 0.026 1439 GGAGTGTTA 2463 QRSALAS 2963 RSDALAR 3463 QSGHLQR 3963 0.075 1440 GGAGTGTTA 2464 QSGALTK 2964 RSDALAR 3464 QSGHLQR 3964 0.035 1441 ATAGCTGGG 2465 RSDHLSR 2965 QSSDLTR 3465 QSGALTQ 3965 0.262 1442 TGCTGGGCC 2466 ERGTLAR 2966 RSDHLTT 3466 DRSHLTK 3966 0.36 1443 TGGAAGGAA 2467 QSGNLAR 2967 RSDNLTQ 3467 RSHHLTT 3967 0.22 1444 TGGAAGGAA 2468 QSGNLAR 2968 RSDNLTQ 3468 RSSHLTT 3968 0.09 1445 TGGAAGCAA 2469 QSGNLAR 2969 RLDNLTA 3469 RSHHLTT 3969 0.182 1446 TGGAAGGAA 2470 QSGNLAR 2970 RLDNLTA 3470 RSSHLTT 3970 0.42 1454 GGAGAGGCT 2471 QSSDLRR 2971 RSDNLAR 3471 QSGHLQR 3971 0.01 1455 CGGGATGAA 2472 QSANLSR 2972 TSGNLVR 3472 RSDHLRE 3972 0.043 1456 GGAGAGGCT 2473 QSSDLRR 2973 RSDNLAR 3473 QRAHLAR 3973 0.016 1457 GCAGAGGAA 2474 QSANLSR 2974 RSDNLAR 3474 QSGSLTR 3974 0.014 1460 TTGGGGGAG 2475 RSDNLAR 2975 RSDHLTR 3475 RADALMV 3975 0.007 1461 GACGAGGAG 2476 RSANLAR 2976 RSDNLTR 3476 DRSNLTR 3976 0.014 1462 CGGGATGAA 2477 QSGNLAR 2977 TSGNLVR 3477 RSDHLRE 3977 0.05 1463 GAGGCTGTT 2478 TTSALTR 2978 QSSDLTR 3478 RSDNLAR 3978 0.003 1464 GACGAGGAG 2479 RSDNLAR 2979 RSDNLTR 3479 DRSNLTR 3979 0.002 1465 CTGGGAGTT 2480 TTSALTR 2980 QSGHLQR 3480 RSDALRE 3980 0.018 1466 CTGGGAGTT 2481 NRATLAR 2981 QSGHLQR 3481 RSDALRE 3981 0.017 1468 GGTGATGTC 2482 DRSALTR 2982 TSGNLVR 3482 MSHHLSR 3982 0.08 1469 GGTGATGTC 2483 DRSALTR 2983 TSGNLVR 3483 TSGHLVR 3983 0.28 1470 GGTGATGTC 2484 DRSALTR 2984 TSGNLVR 3484 QRAHLER 3984 0.156 1471 CTGGTTGGG 2485 RSDHLSR 2985 QSSALTR 3485 RSDALRE 3985 0.09 1472 TTGAAGGTT 2486 TTSALTR 2986 RSDNLTQ 3486 RADALMV 3986 3.22 1473 TTGAAGGTT 2487 TTSALTR 2987 RSDNLTQ 3487 RSDSLTT 3987 0.47 1474 TTGAAGGTT 2488 QSSALAR 2988 RSDNLTQ 3488 RADALMV 3988 1.39 1475 TTGAAGGTT 2489 QSSALAR 2989 RSDNLTQ 3489 RLHSLTT 3989 0.39 1476 TTGAAGGTT 2490 QSSALAR 2990 RSDNLTQ 3490 RSDSLTT 3990 0.305 1477 GCAGCCCGG 2491 RSDHLRE 2991 DRSDLTR 3491 QSGSLTR 3991 2.31 1479 GAAAGTTCA 2492 QSHDLTK 2992 MSHHLTQ 3492 QSGNLAR 3992 37.04 1480 GAAAGTTCA 2493 NKTDLGK 2993 TSGHLVQ 3493 QSGNLAR 3993 62.5 1481 GAAAGTTCA 2494 NKTDLGK 2994 TSDHLAS 3494 RSDELRE 3994 37.04 1482 CCGTGTGAC 2495 DRSNLTR 2995 TSDHLAS 3495 RSDELRE 3995 111.1 1483 CCGTGTGAC 2496 DRSNLTR 2996 MSHHLTT 3496 RSDELRE 3996 20.8 1484 GAAGTGGTA 2497 QSSSLVR 2997 RSDALSR 3497 QSGNLAR 3997 0.01 1485 AAGTGAGCT 2498 QSSDLRR 2998 QSGHLTT 3498 RSDNLTQ 3998 1.537 1486 GGGTTTGAC 2499 DRSNLTR 2999 TTSALAS 3499 RSDHLSR 3999 0.085 1487 TTGAAGGTT 2500 TTSALTR 3000 RSDNLTQ 3500 RLHSLTT 4000 0.188 1488 AAGTGGTAG 2501 QSSDLRR 3001 QSGHLTT 3501 RLDNRTQ 4001 5.64 1490 CTGGTTGGC 2502 RSDHLSR 3002 TSGSLTR 3502 RSDALRE 4002 0.04 1491 AAGGGTTCA 2503 NKTDLGK 3003 DSSKLSR 3503 RLDNRTA 4003 4.12 1492 AAGTGGTAG 2504 RSDNLTT 3004 RSDHLTT 3504 RSDNLTQ 4004 1.37 1493 AAGTGGTAC 2505 RSDNLTT 3005 RSDHLTT 3505 RLDNRTQ 4005 15.09 1494 GGGTTTGAC 2506 DRSNLTR 3006 QRSALAS 3506 RSDHLSR 4006 0.255 1496 TTGGGGGAG 2507 RSDNLAR 3007 RSDHLTR 3507 RSDALTT 4007 0.065 1497 GAGGCTCTT 2508 QSSALAR 3008 QSSDLTR 3508 RSDNLAR 4008 0.007 1498 GAGGTTGAT 2509 QSSNLAR 3009 QSSALTR 3509 RSDNLAR 4009 0.101 1499 GAGGTTGAT 2510 QSSNLAR 3010 TSGALTR 3510 RSDNLAR 4010 0.02 1500 GCAGAGGAA 2511 QSGNLAR 3011 RSDNLAR 3511 QSGSLTR 4011 0.003 1522 GCAATGGGT 2512 TSGHLVR 3012 RSDALTQ 3512 QSGDLTR 4012 0.08

TABLE 6 FINGER (N → C) SEQ SEQ SEQ TRIPLET ID ID ID (5′→3′) F1 NO: F2 NO: F3 NO: AGG RXDHXXQ 4055 ATG RXDAXXQ 4056 CGG RXDHXXE 4057 GAA QXGNXXR 4058 GAC DXSNXXR 4059 DXSNXXR 4059 GAG RXDNXXR 4060 RXSNXXR 4061 RXDNXXR 4060 RXDNXXR 4060 GAT QXSNXXR 4062 TXGNXXR 4064 TXSNXXR 4063 TXGNXXR 4064 GCA QXGSXXR 4065 QXGDXXR 4066 GCC EXGTXXR 4067 GCG RXDEXXR 4068 RXDEXXR 4068 RXDEXXR 4068 RXDTXXK 4069 GCT QXSDXXR 4070 TXGEXXR 4071 QXSDXXR 4070 GGA QXGHXXR 4072 QXAHXXR 4073 GGC DXSHXXR 4074 DXSHXXR 4074 GGG RXDHXXR 4075 RXDHXXR 4075 RXDHXXR 4075 RXDHXXK 4076 GGT TXGHXXR 4077 GTA QXGSXXR 4065 QXATXXR 4078 GTG RXDAXXR 4079 RXDAXXR 4079 RXDAXXR 4079 RXDSXXR 4080 TAG RXDNXXT 4081 TCG RXDDXXK 4082 TGT TXDHXXS 4083 

1. A method for modulating expression of a gene in a cell, wherein the gene comprises a target site, wherein the target site comprises, in a 3′ to 5′ direction, a first (S1), a second (S2) and a third (S3) target subsites, each target subsite having the nucleotide sequence GNN, the method comprising: introducing a polynucleotide encoding a zinc finger protein, into the cell, wherein: (a) the zinc finger protein is a non-naturally-occurring zinc finger protein that has been engineered to bind the target site; and (b) the zinc finger protein comprises first (F1), second (F2), and third (F3) zinc fingers, ordered F1, F2, F3 from N-terminus to C-terminus that bind to (S1), (S2), and (S3), wherein if S1 comprises GAA, F1 comprises the amino acid sequence QRSNLVR (SEQ ID NO:158); if S1 comprises GAG, F1 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130); if S1 comprises GAC, F1 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S1 comprises GAT, F1 comprises the amino acid sequence QSSNLAR (SEQ ID NO:1765); if S1 comprises GGA, F1 comprises the amino acid sequence QSGHLAR (SEQ ID NO:413); if S1 comprises GGG, F1 comprises the amino acid sequence RSDHLAR (SEQ ID NO:127); if S1 comprises GGC, F1 comprises the amino acid sequence DRSHLTR (SEQ ID NO:1506); if S1 comprises GGT, F1 comprises the amino acid sequence QSSHLTR (SEQ ID NO:835); if S1 comprises GCA, F1 comprises QSGSLTR (SEQ ID NO:342); if S1 comprises GCG, F1 comprises RSDDLTR (SEQ ID NO:188); if S1 comprises GCC, F1 comprises ERGTLAR (SEQ ID NO:131); if S1 comprises GCT, F1 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450); if S1 comprises GTA, F1 comprises the amino acid sequence QSGALTR (SEQ ID NO:1398); if S1 comprises GTG, F1 comprises the amino acid sequence RSDALTR (SEQ ID NO:153); if S1 comprises GTC, F1 comprises the amino acid sequence DRSALAR (SEQ ID NO:184); selecting the F2 zinc finger such that it binds to the S2 target subsite, wherein S2 comprises GAA, F2 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801); if S2 comprises GAG, F2 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130); if S2 comprises GAC, F2 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S2 comprises GAT, F2 comprises the amino acid sequence TSGNLVR (SEQ ID NO:1442); if S2 comprises GGA, F2 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287); if S2 comprises GGG, F2 comprises the amino acid sequence RSDHLSR (SEQ ID NO:229); if S2 comprises GGC, F2 comprises the amino acid sequence DRSHLAR (SEQ ID NO:1092); if S2 comprises GGT, F2 comprises the amino acid sequence TSGHLSR (SEQ ID NO:1201); if S2 comprises GCA, F2 comprises the amino acid sequence QSGDLTR (SEQ ID NO:220); if S2 comprises GCG, F2 comprises the amino acid sequence RSDDLQR (SEQ ID NO:1844); if S2 comprises GCC, F2 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417); if S2 comprises GCT, F2 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450); if S2 comprises GTA, F2 comprises the amino acid sequence QSGALAR (SEQ ID NO:3339); if S2 comprises GTG, F2 comprises the amino acid sequence RSDALSR (SEQ ID NO:237); if S2 comprises GTC, F2 comprises the amino acid sequence DRSALAR (SEQ ID NO: 184); and selecting the F3 zinc finger such that it binds to the S3 target subsite, wherein if S3 comprises GAA, F3 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801); if S3 comprises GAG, F3 comprises the amino acid sequence RSDNLTR (SEQ ID NO:231); if S3 comprises GAC, F3 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S3 comprises GAT, F3 comprises the amino acid sequence TSANLSR (SEQ ID NO:377); if S3 comprises GGA, F3 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287); if S3 comprises GGG, F3 comprises RSDHLSR (SEQ ID NO:229); if S3 comprises GGT, F3 comprises the amino acid sequence TSGHLVR (SEQ ID NO:1425); if S3 comprises GCA, F3 comprises the amino acid sequence QSGDLTR (SEQ ID NO:220); if S3 comprises GCG, F3 comprises the amino acid sequence RSDDLTR (SEQ ID NO:188); if S3 comprises GCC, F3 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417); if S3 comprises GCT, F3 comprises the amino acid sequence QSSDLQR (SEQ ID NO:132); if S3 comprises GTG, F3 comprises RSDALTR (SEQ ID NO:153); and if S3 comprises GTC, F3 comprises the amino acid sequence DRSALAR (SEQ ID NO:184); wherein the introduced polynucleotide expresses the zinc finger protein, and zinc finger protein binds to the target site, thereby modulating expression of the gene.
 2. The method of claim 1, wherein the zinc finger protein further comprises a regulatory domain. 